Immunogenic proteoliposomes, and uses thereof

ABSTRACT

An immunogenic proteoliposome containing a transmembrane protein or oligomeric complexes containing such proteins, including viral envelope glycoproteins, in a lipid membrane around an elliptoid or spherical shape. The shape preferably also contains an attractant such as streptavidin or avidin and the lipid membrane contains a moiety that binds to the attractant such as biotin. The immunogenic transmembrane protein is bound to a ligand which is anchored in the shape. Methods for making the immunogenic proteoliposomes are provided uses of the proteoliposome are described, including their use as immunogens to elicit immune reaction, and their use in screening assays, including their use as antigens to screen antibody libraries, as well as for drug screening and the identification of ligands.

[0001] This invention was supported by National Institutes of HealthGrant AI41851 and the government of the United States has certain rightsthereto.

FIELD OF THE INVENTION

[0002] The present invention is directed to immunogenic proteoliposomes,particularly those containing envelope glycoproteins, their constructionand use. Preferably the proteoliposome contains a lentiviral envelopeprotein. Preferably the proteoliposomes are used to elicit animmunogenic response or screen for ligands, including antibodies, smallmolecules, and proteins.

BACKGROUND OF THE INVENTION

[0003] Proteins that are present on the surface of a cell or virus aretypically transmembrane proteins. These proteins include cell surfacereceptors and envelope glycoproteins, and these proteins are involved ina variety of-protein to protein interactions. For example, asdemonstrated with pseudotyped viral particles, the specific envelopeprotein present on a viral surface determines the receptor that thevirus will bind to. Additionally, the three dimensional conformation ofthe protein has an important effect on the particular interaction.However, maintaining a desired conformation can be difficult. Forexample, many receptors, envelope proteins, etc. are the result ofmultimeric formation of individual monomers. Thus, even though eachsubunit may span the membrane only once, the multimeric complex hasseveral membrane-spanning components that could contribute to itsoverall conformational integrity.

[0004] Significant attention has been focused on viruses including theflu virus, herpes virus, retroviruses, lentiviruses, etc., particularlyin the mechanism of infection. Human immunodeficiency virus type 1(HIV-1) and type 2 (HIV-2) are the etiologic agents of acquiredimmunodeficiency syndrome (AIDS), which results from the profounddepletion of CD4-positive lymphocytes in infected individuals(Barre-Sinoussi, F., et al., Science 220:868-71, 1983; Gallo, R. C., etal., Science 224: 500-3, 1984; Fauci, A. S., et al., Ann Intern Med100:92-106, 1984).

[0005] Though great progress has been made in the treatment ofindividuals infected with viruses such as HIV, numerous problems stillremain. For example, treatment typically requires taking cocktails ofmedicines at different times over extended periods of time. The failureto do so can result in seriously undermining the treatment, andultimately results in further progression of the disease. Even whereindividuals follow the treatment protocol, there are many instances ofdisease progression. Moreover, the treatment is extremely costly,effectively rendering it out of reach to many individuals in the UnitedStates, and in much of the rest of the world. There are also otherviruses for which antiviral therapy has not yet been developed.

[0006] Accordingly, the development of alternative methods of dealingwith viral infection, such as HIV infection, is still extremelyimportant.

[0007] One area where a great deal of attention has been extended hasbeen in utilizing viral sub-units to generate immune reactions.Antibodies that neutralize viruses typically do so by inhibiting viralbinding to surface receptors. The major protein found on the surface ofHIV, and therefore a major target for generating neutralizingantibodies, is the envelope glycoprotein, gp120. This protein appears onthe surface of the virion, thus rendering it a prime target for theimmune system.

[0008] Unfortunately, the HIV-1 envelope glycoproteins have proveninefficient in generating antibodies that neutralize the virus,especially those that can neutralize more than a limited number of HIV-1strains (Berman, P. W., et al., J. Infect. Dis. 176: 38497, 1997;Connor, R. I., et al., J. Virol. 72: 1552-76, 1998; Mascola, J. R., etal., J. Infect. Dis. 173: 340-8, 1996; reviewed in Burton, D. R. and D.C. Montefiori, AIDS 11 Suppl. A: S87-98, 1997; Burton, D. R. and J. P.Moore, Nature Med. 4(5 Suppl.) 495-8, 1998; and Wyatt, R. and J.Sodroski, Science 280: 1884-8, 1998). Many of the antibodies elicited bythe envelope glycoproteins are not able to bind efficiently to thefunctional envelope glycoprotein trimer and therefore are devoid ofneutralizing activity (Broder, C. C., et al., Proc. Natl. Acad. Sci. USA91: 11699-703, 1994; Moore, J. P., et al., J. Virol. 69: 101-9, 1995;Moore, J. P., et al., J. Virol. 70: 1863-72, 1996; Parren, P. W., etal., Nature Med. 3:366-7. 1997; Parren, P. W., et al., J. Virol. 72:3512-9, 1998; Wyatt, R., et al., J. Virol. 71: 9722-31, 1997). Thelability of the envelope glycoprotein trimers, conformationalflexibility in the shed gp120 glycoprotein, and the variability andglycosylation of the gp120. surface all appear to contribute to the poorneutralizing antibody responses (reviewed in Montefiori, D. C., et al.,AIDS Res. Human Retroviruses 15: 689-98, 1999; Moore, J., et al., J.Virol. 68: 469-84, 1995; and Wyatt, R., and J. Sodroski, Science 280:1884-8, 1998).

[0009] The entry of primate lentiviruses such as HIV-1 and HIV-2 intotarget cells is mediated by the viral envelope glycoproteins (Wyatt, R.,and J. Sodroski, Science 280: 1884-8, 1998). The mature envelopeglycoproteins on the primate lentivirus are organized into an externalgp120 (gp125 for HIV-2) exterior envelope glycoprotein and the gp41transmembrane envelope glycoprotein (gp36 for HIV-2) (Alan, J. S., etal., Science 228: 10914, 1985; Earl, P. L., et al., J. Virol. 65:2047-55, 1991; Robey, W. G., et al., Science 228: 593-595, 1985;Veronese, F. D., et al., Science 229: 1402-1405, 1985; Wyatt, R., and J.Sodroski, Science 280: 1884-8, 1998). For example, in the infected cell,the HIV-1 envelope glycoprotein is initially synthesized as an 845- to870-amino acid protein, depending upon the viral strain (Earl, P. L., etal, J. Virol. 65: 2047-2055, 1991). N-linked, high-mannose sugars areadded to this primary translation product to result in the gp160envelope glycoprotein precursor (gp140 for HIV-2). Oligomers of gp160form in the endoplasmic reticulum, and several pieces of evidencesuggest that these are trimers. First, X-ray crystallographic studies offragments of the gp41 ectodomain revealed the presence of very stable,six-helix bundles (Chan, D. C., et al., Cell 89: 263-73, 1997; Tan, K.,et al, Proc. Natl. Acad. Sci. USA 94: 12303-8,1997; Weissenhorn, W., etal., Nature 387: 426-30,1997). These structures were composed of atrimeric coiled coil involving N-terminal gp41 α helices, with threeC-terminal gp41 α helices packed into the grooves formed by the threeinner helices. Second, introduction of cysteine pairs at specificlocations in the coiled coil resulted in the formation of intermoleculardisulfide bonds between the gp160 subunits (Farzan, M., et al, J. Virol.72: 7620-5, 1998). The disulfide-stabilized oligomer was shown to be atrimer. Finally, the matrix proteins of HIV-1 and the related simianimmunodeficiency viruses, which interact with the intravirion domains ofthe envelope glycoproteins, crystallize as trimers (Hill, C. P., et al.,Proc. Natl. Acad. Sci. USA 93: 3099-3104, 1996; Rao, Z., et al., Nature378: 743-7, 1995).

[0010] Following oligomerization, the precursor glycoprotein istransported to the Golgi apparatus, where cleavage by a cellularprotease generates the external protein, gp120, and the trans-membraneprotein, gp41 (Alan, J. S., et al., Science 228: 1091-4, 1985; Robey, W.G., et al., Science 228: 593-595, 1985; Veronese, F. D., et a., Science229: 1402-1405, 1985). The gp120 glycoprotein remains associated withthe gp41 glycoprotein through non-covalent, hydrophobic interactions(Helseth, E., et al., J. Virol. 65:2119-23, 1991; Kowalsid, M., et al,Science 237: 1351-1355, 1987). The lability of the gp120-gp41association results in the “shedding” of some gp120 molecules from thetrimer, resulting in non-functional envelope glycoproteins (McKeating,J. A., et al., J. Virol. 65: 852-60, 1991; Willey et al., J. Virol. 68:1029-39, 1994). It has been suggested that these disassembled envelopeglycoproteins result in the generation of high titers ofnon-neutralizing antibodies during natural HIV-1 infection (Burton, D.R., and J. P. Moore, Nat. Med. 4 (5 Suppl.): 495-8, 1998; Moore, J. P.,and J. Sodroski, J. Virol. 70 1863-72, 1996; Parren, P. W., et al., J.Virol. 72: 3512-9). The envelope glycoprotein trimers that remain intactundergo modification of a subset of the carbohydrate moieties to complexforms before transport to the cell surface (Earl, P. L., et al., J.Virol. 65: 2047-55, 1991).

[0011] The mature envelope glycoprotein complex is incorporated from thecell surface into virions, where it mediates virus entry into the hostcell. The gp120 exterior envelope glycoprotein binds the CD4glycoprotein, which serves as a receptor for the virus (Dalgleish, A.G., et al., Nature 312: 763-7, 1984; Klatzmann, D., et al., Nature 312:767-8, 1984; McDougal, J. S., et al, J. Immunol. 137: 2937-44, 1986).Binding to CD4 induces conformational changes in the envelopeglycoproteins that allow gp120 to interact with one of the chemokinereceptors, typically CCR5 or CXCR4 (Alkhatib, G., et al., Science 272:1955-8, 1996; Choe, H., et al., Cell 85: 113548, 1996; Deng, H., et al.,Nature 381: 661-6, 1996; Doranz, B. J., et al., Cell 85: 1149-58, 1996;Dragic, T., et al., Nature 381: 667-73, 1996; Feng, Y., et al., Science272: 872-7, 1996; reviewed in Choe, H., et al., Semin. Immunol. 10:249-57, 1998). The chemokine receptors are seven-transmembrane, Gprotein-coupled receptors, and gp120 interaction with the chemokinereceptors is believed to bring the viral envelope glycoprotein complexnearer to the target cell membrane and to trigger additionalconformational changes in the envelope glycoproteins. Although the exactnature of these changes is unknown, mutagenic data are consistent with arole for the hydrophobic gp41 amino terminus (the “fusion peptide”) inmediating membrane fusion (Cao, J., et al., J. Virol. 67: 2747-55, 1993;Freed, E. O., et al., Proc. Natl. Acad. Sci. USA 87: 4650-4, 1990;Helseth, E., et al., J. Virol. 64: 6314-8, 1990; Kowalsid, M., et al.,Science 237: 1351-5, 1987). It has been suggested that, followinginteraction of the “fusion peptide” with the target cell membrane,formation of the six-helical bundle by the three gp41 ectodomains wouldresult in the spatial juxtaposition of the viral and target cellmembranes (Chan, D. C., et al., Cell 89: 263-73, 1997). Six-helicalbundles have been documented in several viral envelope glycoproteinsthat mediate membrane fusion and virus entry (Bullough, P. A., et al.,Nature 371: 37-43, 1994; Carr, C. M., and P. S. Kim, Cell 73: 823-32,1993; Weissenhorn, W., et al., Proc. Natl. Acad. Sci. USA 95: 6032-6,1998; Weissenhorn, W., et al., Mol. Cell 2: 605-16, 1998). The formationof this energetically stable structure from a different andas-yet-unknown precursor structure is believed to provide the energynecessary to overcome the repulsion between the viral and cellmembranes.

[0012] Initial attempts to generate immune reactions to HIV envelopeglycoproteins have encountered substantial difficulties. For example, itwas discovered that there are numerous regions in the glycoprotein whichrapidly mutate in response to antibodies or drugs directed thereto.These regions also vary significantly from one strain of HIV to another.Accordingly, these regions have been described as variable regions.There are other regions that are conserved among HIV-1, HIV-2 and SIVstrains. Variable regions and conserved regions of gp120 have beenmapped and are well known in the art. In the three-dimensional structureof the protein, these variable regions are typically at the surface, andthus mask the more conserved regions. The variable regions are highlyantigenic, typically generating most of the antibodies seen. It is onlylate in the progression of the disease that antibodies generated to theconserved regions are typically seen. Such antibodies include the F105antibody, the 17b antibody and the 48d antibodies. The amino acidscomprising the epitopes for these antibodies are proximal to each otherin the three-dimensional structure of the protein, but appear distantfrom each other when one looks strictly at a one-dimensional linearamino acid sequence. Such an epitope is referred to as a discontinuousconformational epitope. Furthermore, the amino acids comprising thesediscontinuous conformational epitopes are located in a number ofconserved regions. Numerous variable-region deleted glycoproteins thatexpose these discontinuous conformational epitopes by deleting portionsof the variable regions are disclosed in U.S. Pat. Nos. 5,817,316 and5,858,366.

[0013] Consequently, it is clear that the three-dimensional structure ofthe protein is extremely important in terms of what the immune systemactually sees. Unfortunately, the individual monomers like othermultimeric proteins do not typically form a stable multimer, in thiscase trimeric spikes, that approximate the natural wild typeconfirmation. Thus, generating neutralizing antibodies depends uponstabilizing the three-dimensional, trimeric structure of the envelopeglycoprotein.

[0014] Attempts have been made to stabilize trimers by stabilizinginteractions in the gp41 segment, for example by introducing cysteineresidues. Another approach has been by inserting coiled coils in aportion of the transmembrane protein such as for HIV-1, gp41 or forHIV-2, gp36. Given the importance of being able to make and use suchstable multimers, it is very desirable to have new methods for preparingsuch stable trimers. Particularly so if one wants to use such multimersto elicit an immune response.

[0015] The ability to create new in vitro assays to screen for moleculesthat can interact with a stable multimer such as the envelopeglycoprotein is extremely important.

[0016] The G protein-coupled seven transmembrane segment receptor CXCR4,previously called HUMSTR, LCR-1 or LESTR (Federsppiel et al., 1993;Jazin et al., 1993; Loetscher et al., 1994) has been shown to allow arange of non-human, CD4-expressing cells to support infection and cellfusion mediated by laboratory-adapted HIV-1 envelope glycoproteins (Fenget al., 1996). Other G-protein-coupled seven transmembrane segmentreceptors such as CCR5, CCR3 and CCR2 have been shown to assist cellularentry of other HIV-1 isolates. It is believed that the cellular entryoccurs as a result of the interaction of the external envelopeglycoprotein, e.g., gp120, CD4 and the chemokine receptor. This furtherillustrates the importance of having an in vitro screen for testingmolecules that more closely approximates the wild type env to determinetheir effect on the external env. Thus, the ability to express theenvelope glycoprotein with its three-dimensional structure is extremelyimportant in terms of what the immune system actually sees.

[0017] One of the particular challenges in expressing the env proteinwith its wild type conformation is that it is a transmembrane protein.Transmembrane proteins or integral membrane proteins are amphipathic,having hydrophobic domains that pass through the membrane and interactwith the hydrophobic lipid molecules in the interior of the bilayer, andhydrophilic domains which are exposed to the aqueous environment on bothsides of the membrane (for example, the aqueous environments inside andoutside of the cell). The biological activities of integral membraneproteins (e.g., ligand binding) can be dependent upon the hydrophilicdomains; in some cases, the membrane—spanning regions contribute tofunction.

[0018] It would be desirable to produce, isolate and stabilize inpurified form while retaining their wild-type conformation transmembraneproteins, particularly multimeric proteins, such as gp120, andoligomeric complexes of transmembrane proteins such as gp120/CD4 andgp120/CD4/CCR5. It would be desirable if these proteins could bemaintained in their wild-type conformation for extended periods of timeand under conditions commonly found in vivo. The purification oftransmembrane proteins, including oligomeric complexes, in afunctionally relevant conformation should expedite the ability to elicitimmune reactions to epitopes specifically exposed when the protein(s) isin its the wild-type conformation or in an oligomeric complex, as wellas expedite their use in screening assays to identify antibodies,ligands, and small molecules that bind the transmembrane protein(s).

SUMMARY OF THE INVENTION

[0019] We have now discovered a method for expressing, while maintainingin a wild-type conformation for extended periods of time, transmembraneproteins and oligomeric complexes containing such proteins by formationof proteoliposomes. Preferably, the proteins are envelope proteins.Still more preferably, the proteins are lentiviral proteins.

[0020] The lentiviral protein is preferably from a primate lentivirus,still more preferably a human immunodeficiency virus (HIV-1), e.g. theHIV-1 gp120 or HIV-1 gp160.

[0021] Oligomeric complexes containing lentiviral proteins can includeany lentiviral proteins and any proteins which bind lentiviral proteins.

[0022] Preferably, the proteoliposome contains the purified lentiviralenvelope protein, such as gp120 or gp160. The oligomeric form is atrimer as discussed above. In another preferred embodiment, theproteoliposome contains the lentiviral envelope glycoprotein and acellular receptor such as CD4. In a further embodiment, theproteoliposome contains the lentiviral envelope glycoprotein, CD4, and achemokine receptor, such as CCR5 or CXCR4.

[0023] The proteins expressed in the proteoliposomes are extended by ashort peptide epitope tag, for example the C9 tag, which can berecognized by an antibody (for example, the 1D4 antibody). The tag canbe added to the N-terminus or to the C-terminus of the protein,depending upon the ultimate orientation of the protein in theproteoliposome that is desired. When multiple proteins are expressed inthe same proteoliposome, they can contain the same or different epitopetags. The desired protein is expressed in a cell. Codon optimization maybe used to increase the expression level of the protein. The protein isthen isolated from the cell by a solubilizing agent that maintains theprotein's conformation. Preferably, the solubilizing agent is adetergent. Preferred detergents include alkyl glucopyranosides (such asC8CP, C10-M, C12-M, Cymal-5, Cymal-6 and Cymal-7), alkyl sucroses (suchas HECAMEG), digitonin, CHAPSO, hydroxyethylglucamides (such asHEGA-10), oligoethyleneglycol derivatives (such as C8ES, C8F, andC12E8), dodecylmaltopyranoside, and phenyl polyoxethylenes (such asTriton X-100).

[0024] The detergent-solubilized proteins are then separated from theother cellular debris by capture onto a solid surface (e.g. a sphericalor elliptoid bead). The bead has on its surface an antibody or otherspecific ligand that will capture, orient and concentrate the protein onthe surface of the bead. When multiple proteins are expressed in thesame proteoliposome, the bead can contain the same or differentantibodies to capture, orient and concentrate each protein on thesurface of the bead. The isolated protein is maintained in its wild-typeconformation. Thereafter, it is mixed with a lipid component.Preferably, it also has an attractant for the lipid. For example, thebead can be streptavidin-coated and some lipid component (e.g.biotinyl-DPPE) can be covalently conjugated to biotin. The bead with themixture is then subjected to a known means such as dialysis to form theproteoliposome. The streptavidin-biotin interaction, in this example,helps maintain the lipid layer as the detergent is removed. Theresulting proteoliposome will maintain the integral membrane protein inits native conformation in an isolated and/or purified form for extendedperiods of times.

[0025] These proteoliposomes can be used as immunogens to elicit immunereactions. Alternatively, they can be used to screen antibody librariesfor an antibody. In a preferred embodiment, the stable multimericproteoliposomes can be used as antigens to screen phage display antibodylibraries. Preferably, the immunogenic proteoliposome contains thepurified lentiviral envelope protein, such as gp120 or gp160.

[0026] In another preferred embodiment, the immunogenic proteoliposomecontains the lentiviral envelope glycoprotein and a cellular receptorsuch as CD4.

[0027] In a further preferred embodiment, the immunogenic proteoliposomecontains the lentiviral envelope glycoprotein, CD4, and a chemokinereceptor, such as CCR5 or CXCR4.

[0028] In another preferred embodiment, the immunogenic proteoliposomecontains molecules that can enhance immune responses, for example,costimulatory molecules such as B7-1 or B7-2. It can also containcostimulatory molecules such as LFA-1, ICAM, GM-CSF, or cytokines suchas interleukins or interferons. It can also contain multiplecostimulatory molecules.

[0029] These proteoliposomes can also be used in screening assays suchas drug screening and identifying ligands. In another preferredembodiment, the antigenic proteoliposome contains the lentiviralenvelope glycoprotein and a cellular receptor such as CD4. Preferably,the antigenic proteoliposome contains the purified lentiviral envelopeprotein, such as gp120 or gp160. In a further preferred embodiment, theantigenic proteoliposome contains the lentiviral envelope glycoprotein,CD4, and a chemokine receptor, such as CCR5 or CXCR4.

[0030] These proteoliposomes can also be used to determine the protein'sstructure.

[0031] These proteoliposomes can also be used as a vaccine.

BRIEF DESCRIPTION OF THE DRAWINGS

[0032]FIG. 1 shows a schematic representation of the reconstituted gp160proteoliposomes.

[0033] FIGS. 2A-B show protein composition of PLs.. FIG. 2A shows anSDS-PAGE gel of gp160ΔCT glycoproteins eluted from 5×10⁷ PLs, stainedwith Coomassie blue. Lane 1 shows 1 μg of affinity-purified gp160, lane2 shows protein eluted from gp160ΔCT PLs and lane 3 shows protein elutedfrom beads conjugated with the 1D4 antibody. FIG. 2B shows FACS analysisof PLs stained with the IgGb12 antibody (peak 2) and HIV-1 patient serum(peak 3), compared to anti-human FITC secondary antibody alone (peak 1).

[0034] FIGS. 3A-B show fluorescent microscopic pictures of gp160proteoliposomes. FIG. 3A shows autofluorescence. FIG. 3B shows gp160proteoliposomes reconstituted with a lipid preparation containing 1%DOPE-Rhodamine.

[0035] FIGS. 4A-C show size exclusion chromatography and Western blot ofJR-FL gp160ΔCT glycoproteins eluted from Dynal beads under nativeconditions. 293T cells transiently expressing the JR-FL gp160ΔCTC9-tagged glycoproteins were lysed in CHAPS containing buffer andincubated with Dynal beads conjugated with the 1D4 antibody. Beads werethen washed and incubated in buffer containing 0.2 mM C9 peptide and 0.5M MgCl₂ to elute the gp160ΔCT glycoprotein from the beads. In FIG. 4A,approximately 5 μg of JR-FL gp160ΔCT glycoproteins were analyzed on aSuperdex-200 gel filtration column. In FIG. 4B, eluted fractions werecollected, analyzed by SDS-PAGE under reducing conditions (2% BME) andanalyzed by Western blot with a polyclonal anti-gp120 rabbit serum. InFIG. 4C, eluted fractions were analyzed on a 3-8% SDS-PAGE gradient gelunder non-reducing conditions, and under reducing conditions (2% BME),respectively, and detected by Western blotting using a polyclonalanti-gp120 rabbit serum. Protein bands of apparent molecular weightsconsistent with trimeric gp160ΔCT glycoproteins (T), dimericglycoproteins (D) and monomeric glycoproteins (M) are marked asindicated.

[0036] FIGS. 5A-B shows FACS analysis of the reconstituted PL membrane.FIG. 5A shows occlusion of the 1D4 antibody by lipid membranereconstitution. gp160ΔCT PLs with (peak 2) and without (peak 3) areconstituted membrane were probed with anti-mouse Ig-PE antibody. Peak1 shows staining with the same antibody of non-conjugated beads. In FIG.5B, PLs with a reconstituted membrane containing 1% biotinylated lipid(peak 2) and beads without a reconstituted membrane (peak 1) were probedwith Avidin-FITC.

[0037] FIGS. 6A-B shows binding of the gp41 antibody 2F5 to gp160ΔCTfrom HXBc2 (FIG. 6A) and JR-FL (FIG. 6B) on beads without a membrane(open squares) and fully reconstituted PLs (closed squares). PLs andbeads, respectively, were probed with increasing concentrations of 2F5antibody and anti-human IgG PE antibody and analyzed by FACS. The MFIwas plotted as % maximal MFI at the given antibody concentration.

[0038]FIG. 7 shows binding of a panel of anti-gp120 antibodies and sCD4to YU2 gp160ΔCT glycoprotein expressed on 293T cells compared to YU2gp160ΔCT glycoprotein on PLs. Cells (open circles) and PLs (closedsquares) were incubated with increasing amounts of the indicated humanantibodies, followed by detection with anti-human IgG PE antibody. Fordetection of sCD4 binding to the glycoproteins, the rabbit anti-CD4antibody T45 and anti-rabbit IgG FITC were used. Following staining,samples were analyzed by FACS. The binding of ligands to gp160ΔCT PLswas plotted as % normalized mean fluorescent intensity (MFI) atserially-diluted antibody concentrations. The % normalized MFI valueswere calculated according to the formula:[MFI−MFI(background)]×100/MFI(saturation)−MFI(background). Error barsindicate the range of values obtained for duplicate samples.

[0039]FIG. 8 shows induction of the 17b epitope by sCD4. Binding of the17b antibody to gp160ΔCT glycoprotein on 293T cells and PLs wascompared. Cells and PLs were incubated with and without sCD4 prior tobinding of the 17b antibody and analyzed by FACS. Staining withoutpreincubation with sCD4 was set as 100% and increase of 17b binding isshown for 293T cells (open bars) and PLs (solid bars).

[0040]FIG. 9 shows the effect of proteolytic cleavage of HIV-1 envelopeglycoprotein on ligand binding. Cleavage-competent YU2 gp160ΔCTglycoprotein (closed squares) and cleavage-defective YU2 gp160ΔCTglycoprotein (open circles) was expressed on 293T cells. Cells wereincubated with increasing amounts of the indicated ligands followed bydetection with anti-human IgG-PE. The anti-CD4 polyclonal rabbitantibody T45 and anti-rabbit IgG-FITC was used for the detection of sCD4binding. Normalized MFI was calculated as described for FIG. 7.

[0041]FIG. 10 is a schematic representation of the formation of aparamagnetic CCR5-proteoliposome. The surface of nonporous paramagneticbeads was covalently conjugated with streptavidin and an antibody thatrecognizes the genetically engineered C-terminal C9 tag on CCR5. Theconjugated beads were used to capture the C9-tagged CCR5 from the celllysate. After extensive washing, the beads were mixed withdetergent-solubilized lipid containing approximately 0.1-1% ofBiotinyl-DPPE. During the removal of detergent by dialysis, the lipidbilayer membrane self-assembles around the beads and CCR5 is returned toits native lipid environment.

[0042]FIG. 11 shows quantitation of the lipid acquired by paramagneticCCR5-proteoliposome beads. Approximately 10⁸ 1D4/Streptavidin-conjugatedbeads were reconstituted with CCR5 and different quantities of lipids.The lipid mixtures contained POPC/POPE/DPPA in a 6:3:1 molar ratio, aswell as 1% each (by weight) of biotinyl-DPPE and rhodamine-DOPE. Theintensity of lissamine rhodamine B fluorescence, which was measured byFACS, exhibited a mean value of 20,000 counts. The data points shownrepresent the average of three independent experiments, with standarddeviations indicated. In the inset is the formula by which theapproximate mass of total lipid (m) necessary for complete encapsulationof given number of beads (n) by a single lipid bilayer membrane wascalculated. S is the estimated effective surface of the 2.5-micrometerdiameter Dynal bead. The approximate area occupied by one lipid moleculein the bilayer membrane (P) was considered to be 60 A². NA is Avogadro'snumber and M the average molecular weight of the lipids used formembrane reconstitution.

[0043]FIG. 12 shows binding of the 12G5 antibody toCXCR4-proteoliposomes and to CXCR4-expressing cells.CXCR4-proteoliposomes were prepared as described in the text from cellsexpressing human CXCR4 with a C-terminal C9 tag. The binding of the 12G5antibody, which recognizes a conformation-dependent structure on CXCR4,to the CXCR4-expressing cells and CXCR4-proteoliposomes is shown. Theapparent affinity of the 12G5 antibody for the CXCR4 on theproteoliposome surface is at least as good as that for CXCR4 on cells. Asimilar result was obtained for the conformation-dependent,CXCR4-directed antibody FAB173 (data not shown).

[0044]FIG. 13 shows binding of SDF-1α to CXCR4 on cells andproteoliposoines. Radiolabeled SDF-1α,the natural CXCR4 ligand, wasincubated with either CXCR4-expressing cells or proteoliposomes bearingCXCR4 or CCR5. Unlabeled (cold) SDF-1α was added in increasing amounts,and the amount of radiolabeled SDF-1α bound to the cells orproteoliposomes was measured. The SDF-1α bound with high affinity to theCXCR4-expressing cells and CXCR4-proteoliposomes, but not to theCCR5-proteoliposomes.

[0045] FIGS. 14A-B show FACS analysis of single-chain antibodies.Staining of 293T cells expressing gp160 is represented by the shadedpeaks, and non-expressing control cells is represented by the unshadedpeaks. FIG. 14A shows staining with polyclonal α-gp120 mouse serum andα-mouse-PE. FIG. 14B shows staining with bacterial medium containingphage/single-chain antibodies (1:2 dilution), α-phage mouse IgG andα-mousePE.

[0046]FIG. 15 shows an ELISA of sera from gp160 proteoliposome-immunizedmice and control sera. Prebleed sera was used as negative control sera;PADRE serum refers to mice previously immunized with gp120-PADREglycoproteins that served as a positive control.

[0047]FIG. 16 shows retention of HIV-1 gp120-gp41 association indetergent lysates. The envelope glycoproteins from the 89.6 HIV-1 straincontain a deletion of the gp41 cytoplasmic tail and are tagged at theC-terminus with the C9 peptide epitope, which is recognized by the 1D4antibody. The gp120-gp41 proteolytic cleavage site is intact in thisconstruct. 293T cells were transfected with a plasmid expressing the89.6 envelope glycoproteins, in some cases along with a plasmidexpressing furin (189-192). The cells were lysed in buffer containing 1%CHAPSO, and the lysates incubated with 1D4 antibody-coated beads. Afterwashing, the captured envelope glycoproteins were boiled off the beadsand resolved on a 6% SDS-polyacrylamide gel. The gel was Western blottedand developed with a rabbit anti-gp120 serum and HRP-conjugatedanti-rabbit IgG (left panel). The observed gp120 was precipitated by the1D4 antibody through its association with gp41. The blot was thenstripped and reprobed with the 1D4 antibody and HRP-conjugatedanti-mouse IgG (right panel). Note that, in this example, thecoexpression of furin did not significantly increase the amount ofproteolytically processed envelope glycoproteins captured on the beadsurface, probably because proteolytic cleavage is already very efficientin this context.

[0048]FIG. 17 shows immunization of mice with env-proteoliposomes. In apilot study, four mice per group were immunized intraperitoneally (primeplus two boosts) with gp120 (in Ribi adjuvant), with Env-proteoliposomesalone, or with Env-proteoliposomes in Ribi adjuvant. The ability of thesera to recognize gp120-coated ELISA plates is shown. The ELISA wasdeveloped with horseradish peroxidase-conjugated anti-mouse IgG andoptical density is indicated on the Y axis. Mean values are shown, andless than 30% deviation from the mean was observed in individualanimals. The results indicate that proteoliposomes can elicit antibodyresponses to HIV-1 envelope glycoproteins, and that Ribi adjuvant canenhance those responses.

[0049]FIG. 18 shows that sera from mice immunized withenv-proteoliposomes react efficiently with cell-surface HIV-1 envelopeglycoproteins. Four mice per group were immunized intraperi-toneallywith either Dynal beads coupled to the 1D4 antibody, Env-proteoliposomesalone, or Env-proteoliposomes in Ribi adjuvant. After priming and fourboosts, the sera were tested for the ability to stain cells expressingthe HIV-1 envelope glycoproteins of the same strain as the immunogen.The mean values for mean fluorescence intensity (MFI) are shown for eachgroup of mice.

DETAILED DESCRIPTION OF THE INVENTION

[0050] We have now found a method for expressing transmembrane proteinsand oligomeric complexes containing such transmembrane proteins in largeamounts, purifying and isolating them from other proteins, whilemaintaining them in a wild-type conformation for extended periods oftime. The protein of interest maybe known to be a membrane protein ormay be a putative membrane protein, based upon structure predictionsfrom its distribution of hydrophobic amino acids.

[0051] Preferably, the transmembrane proteins are envelope proteins.Still more preferably, the proteins are lentiviral proteins. Thelentiviral proteins can include, for example, proteins from humanimmunodeficiency virus (HIV), feline immunodeficiency virus (FIC), orvisna virus. Preferably, the transmembrane protein is comprised ofmultimers of the basic unit, such as the trimeric spikes formed by HIV-1or HIV-2 envelope proteins, namely gp160 which cleaves to gp41 and gp120for HIV-1 and gp140 which cleaves to gp125 and gp35 for HIV-2.

[0052] The lentiviral protein is preferably from a primate lentivirus,still more preferably a human immunodeficiency virus (HIV-1), e.g. theHIV-1 gp120 or HIV-1 gp160.

[0053] Oligomeric complexes containing lentiviral proteins can includeany lentiviral proteins and any proteins which bind lentiviral proteins.In another preferred embodiment, the proteoliposome contains thelentiviral envelope glycoprotein and a cellular receptor such as CD4. Ina further embodiment, the proteoliposome contains the lentiviralenvelope glycoprotein, CD4, and a chemoline receptor, such as CCR5 orCXCR4. In another preferred embodiment, the proteoliposome contains thelentiviral envelope glycoprotein and an immune stimulatory molecule suchas B7-1 or B7-2.

[0054] As used herein, an extended period of time is at least 12 hours;preferably at least one day; still more preferably at least one week.Even more preferably an extended period of time is at least one month.Yet more preferably, at least two months.

[0055] Any method of expression may be used to express the desiredtransmembrane protein in a cell, prior to its purification by thepresent invention.

[0056] The list of transmembrane proteins, sometimes also referred to asintegral membrane proteins, is vast. Transmembrane proteins may crossthe membrane only once or over twenty times. Many transmembrane proteinsassociate with other transmembrane proteins to form larger complexes.Such complexes may be comprised of two identical subunits (such ashomodimers) or two different protein subunits (such as heterodimers).There are examples of even larger complexes of three (sodium ionchannel, Na⁺/K⁺ ATPase), four (aquaporin), five (cation channels ofnicotinic receptors, anion channels of glycine receptors) or morephotoreaction center, mitochondrial respiratory chain) homologous orheterologous subunits.

[0057] Transmembrane proteins contribute to a wide variety of cellularfunctions, including the transport of molecules and ions into or out ofcells, cell recognition, cell-to-cell communication, and cell adhesion.One simple way to classify transmembrane proteins is by their number oftransmembrane domains (Table 1).

[0058] The group of transmembrane proteins that only cross the membraneonce (also known as single-pass proteins) is particularly diverse bothstructurally and functionally. This class includes envelopeglycoproteins and a large number of cell surface receptor proteins. Forexample, the EGF receptor binds epidermal growth factor, which leads toactivation of the receptor's tyrosine linase activity. Other examples ofsingle-pass transmembrane proteins include the integrins and cadherins,which function in cell-cell communication via binding to extracellularmolecules.

[0059] Other examples of viral envelope proteins include, for example,envelope proteins from filoviruses (such as Ebola virus),orthomyxoviruses (such as influenza virus), VSV-G, s alpha viruses (suchas Semliki forest virus and Sindbis virus), arena viruses (such aslymphocytic choriomeningitis virus), flaviviruses (such as tick-borneencephalitis virus and Dengue virus), rhabdoviruses (such as vesicularstomatitis virus and rabies virus), Moloney leukemia virus, HSV,VZV,Mumps virus, Rhinoviruses, Measles, Rubella, Arbovirus, Enteroviruses(such as Polio, Coxsackie, Echoviruses), Polio virus, Coxsackie B, A &Echovirus, Rhinoviruses, Hepatitis viruses, Norwalk virus, Astroviruses,Togavirus, Alphaviruses, Pestiviruses, Coronavirus, Parainfluenza, Mumpsvirus, Measles virus, Respiratory Syncytial Virus (RSV), Bunyaviridae,Reoviridue, Reoviruses, Rotaviruses, HTLV, Polyomaviruses,Papillomaviruses, Adenoviruses, Parvoviruses, EBV, CMV, Varicella Zostervirus, herpes viruses, and Pox viruses.

[0060] Another large class of cell surface receptors is the G-proteincoupled receptors (GPCRs), which span the membrane seven times. Unlikemany of the single-pass receptors, these proteins do not have enzymaticactivity themselves but instead are functionally linked to signalingproteins known as G proteins. The chemokine receptor CCR5 that serves asthe principal coreceptor for HIV-1 is a typical example of a Gprotein-coupled receptor.

[0061] Other well studied members of this class include transducin,which senses light, and the acetylcholine receptor, which bindsneurotransmitter at neuronal synapses. # TM Protein Class Specificexample domains Reference Receptor guanylyl cyclases Sperm Reactreceptor 1 MBOC pp. 759-60 Receptor tyrosine kinases EGF receptor 1 MBOCpp. 759-60 Protein tyrosine phosphatases CD45 1 MBOC pp. 768 IntegrinsAlpha, beta chains 1 MBOC pp. 996-7 Cadherins E-cadherin 1 MBOC pp.996-7 Chemotaxis receptors 2 MBOC pp. 775-6 Some potassium channels KcsK channel 2 Doyle et al. Connexins 4 MBOC pp. 959 Photosyntheticreaction center L, M subunits 5 MBOC pp. 498 Some ABC transporters 6Reimann & Ashcroft Voltage-gated K⁺ channels Shaker 6 Reimann & AshcroftG-coupled receptors Transducin 7 Chemokine receptors 7 Acetylcholinereceptor 7 Ion pumps Ca⁺⁺ pump catalytic 10 MBOC pp. 516 subunitNa⁺−K⁺pump catalytic 10 MBOC pp. 516 sub. CIC channels CIC-1 of skeletalmuscle 11 Valverde ABC transporters MDR ATPase 12 MBOC pp. 522 Peptidepump 12 MBOC pp. 522 CFTR 12 MBOC pp. 522 Anion transporters Band 3protein 14 MBOC pp.

[0062] Sequences of these proteins are widely available in theliterature and from computer databases such as Genbank. Thus, one canreadily obtain the gene encoding a particular protein of interest. Thisgene can be expressed by any known means. These include creating anexpression cassette, where the gene is operably linked to a promoter.Other enhancing elements are known and may also be used. The codons usedto synthesize the protein of interest may be optimized, converting themto codons that are preferentially used in mammalian cells. Optimalcodons for expression of proteins in non-mammalian cells are also known,and can be used when the host cell is a non-mammalian cell (for example,insect cells, yeast cells, bacteria).

[0063] The gene is then introduced into a cell for the expression byknown means. For example, they can include vectors, liposomes, nakedDNA, adjuvant-assisted DNA, gene gun, catheters, etc. Vectors includechemical conjugates, plasmids, phage, etc. The vectors can bechromosomal, non-chromosomal or synthetic. Commercial expression vectorsare well known in the art, for example pcDNA3.1, pcDNA4 HisMax, pACH,pMT4, PND, etc. Promoters that can be used to express the gene are wellknown in the art. The promoter chosen are selected based upon the hostcell which the protein is expressed in. These include cytomegalovirus(CMV) intermediate early promoter, a viral LTR such as the Rous sarcomavirus LTR, HIV-LTR, HTLV-1 LTR, the simian virus 40 (SV40) earlypromoter, E. coli lac V5 promoter and the herpes simplex TK viruspromoter.

[0064] Preferred vectors include viral vectors, fusion proteins andchemical conjugates. Retroviral vectors include Moloney murine leukemiaviruses. Other vectors include pox vectors such as orthopox or avipoxvectors, herpesvirus vectors such as a herpes simplex I virus (HSV)vector (Geller, A. I. et al., (1995), J. Neurochem, 64: 487; Lim, F., etal., (1995) in DNA Cloning: Mammalian Systems, D. Glover, Ed., OxfordUniv. Press, Oxford England; Geller, A. I. et al. (1993), Proc. Natl.Acad. Sci.: U.S.A. 90:7603; Geller, A. I., et al., (1990) Proc Natl.Acad. Sci USA 87:1149), adenovirus vectors (LeGal LaSalle et al. (1993),Science, 259:988; Davidson, et al. (1993) Nat. Genet 3: 219; Yang, etal., (1995) J. Virol. 69: 2004) and adeno-associated virus vectors(Kaplitt, M. G., et al. (1994) Nat. Genet. 8: 148).

[0065] The particular vector chosen will depend upon the host cell used.

[0066] The introduction of the gene into the host cell can be bystandard techniques, e.g. infection, transfection, transduction ortransformation. Examples of modes of gene transfer include e.g., nakedDNA, CaPO4 precipitation, DEAE dextran, electroporation, protoplastfusion, lipofection, cell microinjection, and viral vectors.

[0067] An antigenic tag may be inserted in the protein to assist in itspurification and in orienting the protein on the solid surface.Preferably, the tag is present at either the N-terminal end or theC-terminal end of the protein. The tag is preferably 6 to 15 amino acidsin length, still more preferably about 6 to 9 amino acids. The tag isselected and its coding sequence inserted into the gene encoding theprotein in a manner not to affect the overall conformation or functionof the protein. Tags can include HA, polyoma, C9, FLAG, etc.

[0068] The integral membrane protein expressing cell is then lysed in abuffer with the appropriate detergent and protease inhibitors so theprotein can be separated from other cellular debris by conventionalmeans without harming the protein.

[0069] In general, due to their amphipathic properties, transmembraneproteins can be solubilized only by agents that disrupt hydrophobicassociations and destroy the membrane's lipid bilayer. The agentstypically used are small amphipathic molecules which tend to formmicelles in water. Preferably, the agent is a detergent. When mixed withmembranes, the hydrophobic regions of the detergent bind to thetransmembrane domain of proteins, displacing the lipid molecules. Thepolar ends of detergents can either be charged (ionic) or uncharged(non-ionic). Although integral membrane proteins can be maintained in anative conformation in a detergent solution, over time many suchsolubilized proteins undergo denaturation and aggregation.

[0070] When a detergent is removed from a transmembraneprotein-detergent complex in the absence of phospholipid, the membraneprotein molecules usually denature, aggregate and precipitate out ofsolution. If, however, the purified protein is mixed with phospholipidbefore the detergent is removed, the active protein can insert into thelipid bilayer formed by the phospholipids. In this manner, functionallyactive membrane proteins can be reconstituted from purified components.An integral membrane protein properly reconstituted into its nativelipid environment is stable for extended periods of time.

[0071] Additionally, a critical factor for maintaining a functionalconformation of a membrane protein during its purification is the choiceof detergent used to solubilize the protein. The detergent best suitedfor a given membrane protein is typically determined empirically. If theprotein has been investigated previously, the literature will indicatesuccessful detergents. Moreover, one can rely upon the results obtainedwith related proteins to determine detergents that will be successfulwith other proteins. Thus, research on a related protein indicates thetype of detergent most likely to extract the protein in an active form.

[0072] Detergents can be generally classed, depending upon the nature oftheir polar end, into three groups: non-ionic, zwitterionic, and ionic.Strong ionic detergents (such as SDS) can solubilize most membraneproteins, but tend to unfold the protein in the process, making themless useful for reconstituting active conformations. In general, mildernon-ionic detergents are preferred.

[0073] Detergents recommended for gentle solubilization of membraneproteins include alkyl glucopyranosides (such as C8-GP and C9-GP), alkylthio-glucopyranosides (such as C8-tGP, C10-M, C12-M, Cymal-5, Cymal-6,and Cymal-7), alkyl sucroses (such as HECAMEG), CHAPSO, digitonin,hydroxyethylglucamides (such as HEGA-10), oligoethyleneglycolderivatives (such as C8E5, C8En, and C12E8), dodecylmaltopyranoside, andphenyl polyoxyethylenes (such as Triton X-100).

[0074] Preferred detergents include alkyl thioglucopyranosides,dodecylmaltopypanoside and phenyl polyoxyethydenes. More preferably,Cymal-5, Cymal-6, Cymal-7, HEGA-10, digitonin, CHAPSO,dodecylmaltopyranoside, and Triton X-100. Still more preferably Cymal-5,Cymal-6, Cymal-7, and dodecylmaltopyranoside.

[0075] Commercial kits are also available to assist in choosing adetergent appropriate for a given membrane protein. For example, bothAnatrace and Calbiochem offer a variety of kits containing mixtures ofdifferent detergents.

[0076] There are many known instances of detergents which have beensuccessfully used to purify functionally active membrane proteins. Forexample, decylmaltoside was used to purify the K⁺ channel (Ksc K⁺) fromStreptomyces lividans, allowing its structure to be determined by X-raycrystallography (Doyle et al., Science (1998)280: 69-77). Cymal-5,Cymal-6, Cymal-7, and dodecylmaltopypanoside are preferred detergentsfor GCPRs, more preferably for chemokine receptors (Mirzabekov, T. etal. (1999), J. Biol. Chem. 274: 28745-50).

[0077] The cleared cell lysate containing all solubilized membraneproteins and other water-soluble cellular proteins can be separated fromthe other cellular debris by conventional means. For example using highspeed centrifugation, such as 150,000×g. Antibodies directed against theepitope tag on the protein of interest are used to capture this proteinfrom the cell lysate onto the solid support (e.g., beads). After bindingof the solubilized integral membrane protein to the antibodiesimmobilized on the solid support, the solid support is washed.Thereafter the purified detergent-protein mixture is formed into aproteoliposome as described below.

[0078] The proteoliposome comprises a spherical or elliptoid shape suchas a bead or other pellet. Preferably, the bead or pellet is at leastabout 15% the size of a eukaryotic cell; still more preferably it is atleast about 20% the size of such a cell; and even more preferably it isat least about 25% the size of such a cell. The shape isthree-dimensional so that it can be coated on all sides. However, therecan be substantial variability in the exact shape used. The exact shapechosen will depend upon the way the proteoliposome is being used. Thus,in some embodiments flakes are preferable to beads, e.g., as animmunogen, in others, a thicker ellipsoid can be preferable.

[0079] Any lipid or lipid mixture that supports membrane reconstitutioncan be used. Preferred lipids include1-Palmitoyl-2-Oleoyl-sn-Glycero-3-Phosphocholine (POPC),1-Palmitoyl-2-Oleoyl-sn-Glycero-3-Phosphoethanolamine (POPE),Dimyristoylphosphatidic acid (DMPA) and Cholesterol. Preferably amixture of these lipids is used, for example at molar concentrations45:25:20:10. The lipid mixture can be dried in a 2-ml polyethylene tubeunder a vacuum until all of the solvent is removed. PBS can be added tothe tube and a liposomal solution can be obtained by ultrasonication for5 minutes in an ice bath using an Ultrasonic Processor (Heat Systems,Inc., Farmingdale, N.Y.).

[0080] The spherical or elliptoid shape, e.g. bead, is preferably alsocoated with a substance that will help attract and anchor a lipid layer.For example, one can use a compound such as streptavidin or avidin tocoat the spherical or elliptoid shape such as a bead and add a smallamount of biotinylated lipid to the lipid mixture. For example, one canuse a head group-modified synthetic lipid, such asdipalmitoylphosphoethanolamine-N-Biotinyl (3iotinyl-DPPE) ordioleoylphosphoethanolamine-lissamine Rhodamine B (Rho-DOPE) in solutionwith lipids. Such a mixture will form a strong uniform coating with, forexample, a streptavidin coated-bead. Liposomal solutions of the headgroup-modified synthetic lipids1,2-dioleyl-sn-glycero-3-phosphoethanolamine-n-(biotinyl)(Biotinyl-DOPE) and dioleoylphosphoethanolamine-lissamine rhodamine B(Rho-DOPE), at a final concentration of 1 mg/ml, can be preparedseparately using the same protocol as described above for thenon-labeled lipid solutions.

[0081] The spherical or elliptoid shape (such as a bead) will also havean anchor ligand such as an antibody bound to it that will specificallybind either the antigenic tag or a known specific portion of theintegral membrane protein that is to be bound to the bead, therebyorienting the protein. The lipid solution containing biotinylated lipidis added to the beads with the captured protein of interest. Thereafter,the detergent is slowly removed by known means. For example, bydialysis, for e.g., at least 24 hours. The resulting integral membraneprotein-containing proteoliposome is stable for an extended period oftime. As used herein, an extended period of time means at least 12hours; still more preferably at least one day; even more preferably atleast one week; still more preferably at least one month; and even morepreferably at least two months. Not only will the protein retain itsconformation in these proteoliposomes for long periods of time, but itwill do so under a wide range of conditions, such as pH and temperature.

[0082] Preferably the spherical or elliptoid surface that is used is amagnetic bead. Magnetic beads are well known in the art and can beobtained commercially. For example tosylactivated Dynabeads® M-(Bikker,J. A., Trumpp-Kallmeyer, S., and Humblet, C. (1998) J. Med. Chem. 41,2911-2927)0 (Dynal, Inc., Lake Success, N.Y.). These are particularlyuseful in assisting in the purification of the protein. One can use suchproteoliposomes as intermediates and transfer the stabilizedproteoliposome to another surface. For example, a flake. When using theproteoliposome for injection into an individual, it is preferable thatthe surface is made of a biodegradable material.

[0083] While the proteoliposome will typically contain only the integralmembrane protein of interest, there are instances where one may want touse more than one protein. For example, the chemoline receptor CCR5 isknown to cooperate with the single transmembrane-spanning protein, CD4,in interacting with the HIV gp120 protein. Thus, one can prepareproteoliposomes containing CD4 as well as the envelope glycoprotein. Inanother embodiment one can have both CCR5 and CD4 as well as theenvelope glycoprotein. This can readily be done by tagging the proteinswith the same epitope tag at the C-terminus and preparing beads with theappropriate tag-reactive antibody. Alternatively, the proteins can betagged with different tags and one can prepare beads having mixtures ofdifferent antibodies. This would allow one to vary the ratios of the twoproteins in the proteoliposome.

[0084] These stabilized proteoliposomes can be used in a variety ofdifferent methods.

[0085] One can obtain high concentrations of the protein on the bead. Inthis manner one can use the proteoliposome as an immunogen to obtainantibodies to the native confirmation of the protein. One can use theproteoliposomes to obtain antibodies to different epitopes exposedduring different conformations of a protein. For example, one proteinmay assemble into several different multimeric complexes, depending forexample on the availability of different binding partners.Proteoliposomes carrying different complexes can be used as immunogens,thus generating antibodies to different epitopes on a single proteinwhich are differentially exposed depending on its binding to otherproteins.

[0086] The immunogenic proteoliposomes can be used to generate and alsoto identify a range of antibodies. For example, antibodies to gp120 andgp41 (or gp135 and gp35). For example, antibodies that affect theinteraction with the receptor binding sites can be directly screenedfor, for instance by using a direct binding assay. For example, one canuse a radioactive or fluorescent marker to label the gp120proteoliposome and add soluble CD4, or more preferably a proteoliposomecontaining CD4. There are various soluble CD4s known in the artincluding a two-domain (D1D2 sCD4) and a four-domain version. The CD4proteoliposomes can be added to medium containing the gp120proteoliposome and an antibody that will block binding between the twoproteoliposomes can be screened for. In another example, theproteoliposome can contain both gp120 and CD4 and you can look atinteractions with CCR5. Alternatively, when using a derivative from a Tcell tropic gp120 one would use a proteoliposome containing CXCR4.Binding can then be directly measured. The antibody of interest can beadded before or after the addition of the labeled proteoliposome and theeffect of the antibody on binding can be determined by comparing thedegree of binding in that situation against a base line standard withthat proteoliposome, in the absence of the antibody.

[0087] A preferred assay uses the labeled proteoliposome, for examplecontaining a gp120 trimer derived from an M-tropic strain such as JR-FL,iodinated using for instance solid phase lactoperoxidase (in one examplehaving a specific activity of 20 μCi/μg). The proteoliposome containingthe chemokine receptor in this example would contain CCR5. Soluble CD4could be present.

[0088] gp120 Derivatives

[0089] The proteoliposome can contain a variety of envelope glycoproteinderivatives.

[0090] In one embodiment, the envelope glycoprotein trimer is composedof variable region-deleted gp120 or gp125 such as described in U.S. Pat.Nos. 5, 858,366 and 5, 817,316. For example, the conformational gp120portion should contain a sufficient number of amino acid residues todefine the binding site of the gp120 to the chemokine receptor (e.g.sequences from the fourth conserved region and from the V3 loop) and asufficient number of amino acids to maintain the conformation of thepeptide in a conformation that approximates that of wild-type gp120bound to soluble CD4 with respect to the chemokine receptor bindingsite. In other embodiments the V3 loop can be removed to remove maskingamino acid residues. In order to maintain the conformation of thepolypeptide one can insert linker residues that permit potential turnsin the polypeptides structure. For example, amino acid residues such asGly, Pro and Ala. Gly is preferred. Preferably, the linker residue is assmall as necessary to maintain the overall configuration. It shouldtypically be smaller than the number of amino acids in the variableregion being deleted. Preferably, the linker is 8 amino acid residues orless, more preferably 7 amino acid residues or less. Even morepreferably, the linker sequence is 4 amino acid residues or less. In onepreferred embodiment the linker sequence is one residue. Preferably, thelinker residue is Gly.

[0091] In one preferred embodiment, the gp120 portion also contains aCD4 binding site (e.g. from the C3 region residues 368 and 370, and fromthe C4 region residues 427 and 457). The chemokine binding site is adiscontinuous binding site that includes portions of the C2,,C3, C4 andV3 regions. By deletion of non-essential portions of the gp120polypeptide—such as deletions of portions of non-essential variableregions (e.g. V1/V2) or portions in the constant regions (e.g. C1, C5)one can increase exposure of the CD4 binding site. Another embodiment isdirected to a gp120 portion containing a chemokine binding site.Similarly, by deleting the non-essential portions of the protein one canincrease exposure of the chemokine binding site. The increased exposureenhances the ability to generate an antibody to the CD4 receptor orchemookine receptor, thereby inhibiting viral entry. Removal of theseregions is done while requiring the derivative to retain an overallconformation approximating that of the wild-type protein with respect tothe native gp120 binding region, e.g. the chemoline binding region whencomplexed to CD4. In addition, one can remove glycosylation sites thatare dispensable for proper folding. Maintaining conformation can beaccomplished by using the above-described linker residues that permitpotential turns in the structure of the gp120 derivative to maintain theoverall three-dimensional structure. Preferred amino acid residues thatcan be used as linker include Gly and Pro. Other amino acids can also beused as part of the linker, e.g. Ala. Examples on how to prepare suchpeptides are described more fully in Wyatt, R, et al., J. Virol. 69:5723-5733, 1995; Thali, M., et al., J. Virol. 67: 3978-3988, 1993; andU.S. Pat. Nos. 5,858,366 and 5,817,316, which are incorporated herein byreference.

[0092] An alternative gp120 derivative is one wherein the linkers usedresult in a conformation for the derivative so that the discontinuousbinding site with the chemokine receptor approximates the conformationof the discontinuous binding site for the chemokine receptor in thewild-type gp120/CD4 complex. These derivatives can readily be made bythe person of ordinary skill in the art based upon the above describedmethodologies and screened in the assays shown herein to ensure thatproper binding is obtained.

[0093] In one embodiment, at least one sugar addition site is deleted.Preferably the sugar addition site is near a conformational epitope.This can be accomplished by known means. For example, the amino acid canbe deleted. In one embodiment that deleted amino acid can be replaced byanother residue that will not form a sugar addition site.

[0094] In a preferred embodiment, multiple sugar addition sites can bedeleted. In a still more preferred embodiment the sugar addition sitescan be deleted from the variable loop deleted monomers.

[0095] Generating Antibodies

[0096] These immunogenic proteoliposomes can be used to generate animmune reaction in a host by standard means. For example one canadminister the proteoliposome in adjuvant.

[0097] The proteoliposome is preferably administered with an adjuvant.Adjuvants are well known in the art and include aluminum hydroxide, Ribiadjuvant, etc. Preferably the proteoliposome is comprised ofbiodegradable material.

[0098] One can administer these proteoliposomes to individuals by avariety of means. For immunization purposes, intradermal, subcutaneous,intramuscular and mucosal administration can be used.

[0099] The proteoliposomes when used for administration are preparedunder aseptic conditions with a pharmaceutically acceptable carrier ordiluent.

[0100] Doses of the pharmaceutical compositions will vary depending uponthe subject and upon the particular route of administration used.Dosages can range from 0.1 to 100,000 μg/kg a day, more preferably 1 to10,000 μg/kg.

[0101] Routes of administration include oral, parenteral, rectal,intravaginal, topical, nasal, direct injection, etc.

[0102] An exemplary pharmaceutical composition is a therapeuticallyeffective amount of an oligomer, antibody etc., that for example affectsthe ability of the receptor to facilitate HIV infection, or that caninduce an immune reaction, thereby acting as a prophylactic immunogen,optionally included in a pharmaceutically-acceptable and compatiblecarrier. The term “pharmaceutically-acceptable and compatible carrier”as used herein, and described more fully below, includes one or morecompatible solid or liquid filler diluents or encapsulating substancesthat are suitable for administration to a human or other animal. In thepresent invention, the term “carrier” thus denotes an organic orinorganic ingredient, natural or synthetic, with which the molecules ofthe invention are combined to facilitate application. The term“therapeutically-effective amount” is that amount of the presentpharmaceutical composition which produces a desired result or exerts adesired influence on the particular condition being treated. Forexample, the amount necessary to raise an immune reaction to provideprophylactic protection. Typically when the composition is being used asa prophylactic immunogen at least one “boost” will be administered at aperiodic interval after the initial administration. Variousconcentrations may be used in preparing compositions incorporating thesame ingredient to provide for variations in the age of the patient tobe treated, the severity of the condition, the duration of the treatmentand the mode of administration.

[0103] In one preferred method of immunization one would prime with aproteoliposome containing variable loop deleted gp120 trimer, and thenboost with a proteoliposome containing a gp120 trimer that more closelyapproximates the wild type viral glycoprotein until at least one finalboost with proteoliposomes containing the stabilized wild type trimer.For example, if multiple variable regions and sugar addition sites aredeleted from the priming trimer, the next boost will be with a trimerwhere more variable region amino acids are present and/or sugar additionsites present. Each boost will get closer to the wild type configurationuntil that configuration is reached.

[0104] Doses of the pharmaceutical compositions of the invention willvary depending on the subject and upon the particular route ofadministration used. Dosages can range from 0.1 to 100,000 μg/kg perday, more preferably 1 to 10,000 μg/kg. By way of an example only, anoverall dose range of from about, for example, 1 microgram to about 300micrograms might be used for human use. This dose can be delivered atperiodic intervals based upon the composition. For example on at leasttwo separate occasions, preferably spaced apart by about 4 weeks. In theembodiment where the prime is the proteoliposome containing the variableloop deleted gp120 trimers, with the boost of proteoliposomes containingnative gp120, or native gp120, it is presently preferred to have aseries of at least 2 boosts, preferably 3 to 5 boosts spread out over ayear. Other compounds might be administered daily. Pharmaceuticalcompositions of the present invention can also be administered to asubject according to a variety of other, well-characterized protocols.For example, certain currently accepted immunization regimens caninclude the following: (i) administration times are a first dose atelected date; a second dose at 1 month after first dose; and a thirddose at a subsequent date, e.g., 5 months after second dose. See ProductInformation, Physician's Desk Reference, Merck Sharp & Dohme (1990), at144243. (e.g., Hepatitis B Vaccine-type protocol); (ii) for example withother vaccines the recommended administration for children is first doseat elected date (at age 6 weeks old or older); a second dose at 4-8weeks after first dose; a third dose at 4-8 weeks after second dose; afourth dose at 6-12 months after third dose; a fifth dose at age 4-6years old; and additional boosters every 10 years after last dose. SeeProduct Information, Physician 's Desk Reference, Merck Sharp & Dohme(1990), at 879 (e.g., Diphtheria, Tetanus and Pertussis-type vaccineprotocols). Desired time intervals for delivery of multiple doses of aparticular composition can be determined by one of ordinary skill in theart employing no more than routine experimentation.

[0105] Antibodies

[0106] The term “antibodies” is meant to include monoclonal antibodies,polyclonal antibodies and antibodies prepared by recombinant nucleicacid techniques that are selectively reactive with polypeptides encodedby nucleotide sequences of the present invention. The term “selectivelyreactive” refers to those antibodies that react with one or moreantigenic determinants on e.g. gp120 and do not react with otherpolypeptides. Antigenic determinants usually consist of chemicallyactive surface groupings of molecules such as amino acids or sugar sidechains and have specific three dimensional structural characteristics aswell as specific charge characteristics. Antibodies can be used fordiagnostic applications or for research purposes, as well as to blockbinding interactions.

[0107] For preparation of antibodies directed toward the immunogenicproteoliposomes, any technique that provides for the production ofantibody molecules may be used.

[0108] For example, mice can be immunized twice intraperitoneally withapproximately 50 micrograms of proteoliposome immunogen per mouse. Serafrom such immunized mice can be tested for antibody activity byimmunohistology or immunocytology on any host system expressing suchpolypeptide or against another proteoliposome or by ELISA with theexpressed polypeptide. For immunohistology, active antibodies of thepresent invention can be identified using a biotin-conjugated anti-mouseimmunoglobulin followed by avidin-peroxidase and a chromogenicperoxidase substrate. Preparations of such reagents are commerciallyavailable; for example, from Zymed Corp., San Francisco, Calif. Micewhose sera contain detectable active antibodies according to theinvention can be sacrificed three days later and their spleens removedfor fusion and hybridoma production. Positive supernatants of suchhybridomas can be identified using the assays described above and by,for example, Western blot analysis.

[0109] Another method for preparing antibodies is by using hybridomamRNA or splenic mRNA as a template for PCT amplification of such genes[Huse, et al., Science 246:12176 (1989)]. For example, antibodies can bederived from murine monoclonal hybridomas [Richardson, J. H., et al.,Biochem and Biophys Res Comm. 197: 422427 (1993); Mhashilkar, A. M., etal., EMBO J. 14:1542-1551 (1995)]. These hybridomas provide a reliablesource of well-characterized reagents for the construction of antibodiesand are particularly useful when their epitope reactivity and affinityhas been previously characterized. Another source for such constructionincludes the use of human monoclonal antibody producing cell lines[Marasco, W. A., et al., Proc. Natl. Acad. Sci. USA 90:7889-7893 (1993);Chen, S. Y., et al., Proc. Natl. Acad. Sci. USA 91:5932-5936 (1994)].Another example includes the use of antibody libraries such as phagedisplay technology to construct new antibodies against differentepitopes on a target molecule [Burton, D. R., et al., Proc. Natl. Acad.Sci. USA 88:10134-1-137 (1991); Hoogenboom, H. R., et al., Immunol. Rev.130:41-68 (1992); Winter, G., et al., Ann. Rec. Immunol 12:433-355(1994); Marks, J. D., et al., J. Biol. Chem. 267:16007-16010 (1992);Nissim, A., et al., EMBO J. 13:692-698 (1994); Vaughan, T. J., et al.,Nature Bio. 14:309-314 (1996); Marks, C., et al., New Eng. J. Med. 335:730-733 (1996)]. For example, very large naive human sFV libraries havebeen and can be created to offer a large source of rearranged antibodygenes against a plethora of target molecules. Smaller libraries can beconstructed from individuals with autoimmune disorders [Portolano, S,.et al., J. Immunol. 151:2839-2851 (1993); Barbas, S. M., et al., Proc.Natl. Acad. Sci. USA 92:2529-2533 (1995)] or infectious diseases[Barbas, C. F., et al., Proc. Natl. Acad. Sci. USA 89:9339-9343 (1992);Zebedee, S. L., et al., Proc. Natl. Acad. Sci. USA 89:3175-3179 (1992)]in order to isolate disease specific antibodies. Thereafter one can usethese proteoliposomes to screen such libraries to identify the desiredantibodies.

[0110] Other sources include transgenic mice that contain a humanimmunoglobulin locus instead of the corresponding mouse locus as well asstable hybridomas that secrete human antigen-specific antibodies[Lonberg, N., et al., Nature 368:856-859 (1994); Green, L. L., et al.,Nat. Genet. 7:13-21 (1994)]. Such transgenic animals provide anothersource of human antibody genes through either conventional hybridomatechnology or in combination with phage display technology. In vitroprocedures to manipulate the affinity and find specificity of theantigen binding site have been reported including repertoire cloning[Clackson, T., et al., Nature 352: 624-628); marks, J. D., et al., J.Mol. Biol. 222: 581-597 (1991); Griffiths, A. D., et al., EMBO J. 12:725-734 (1993)], in vitro affinity maturation [Marks, J. D., et al.,Biotech 10: 779-783 (1992); Gram, H., et al., Proc. Natl. Acad. Sci. USA89: 3576-3580 (1992)], semi-synthetic libraries [Hoogenboom, H. R.,supra; Barbas, C. F., supra; Akamatsu, Y., et al., J. Immunol. 151:4631-4659 (1993)] and guided selection [Jespers, L. S. et al., Bio Tech12: 899-902 (1994)]. Starting materials for these recombinant DNA basedstrategies include RNA from mouse spleens [Clackson, t., supra] andhuman peripheral blood lymphocytes [Portolano, S., et al., supra;Barbas, C. F., et al., supra; Marks, J. D., et al., supra; Barbas, C.F., et al., Proc. Natl. Acad. Sci. USA 88: 7978-7982 (1991)] andlymphoid organs and bone marrow from HIV-1-infected donors [Burton, D.R., et al., supra; Barbas, C. F., et al., Proc. Natl. Acad. Sci. USA89:9339-9343 (1992)].

[0111] For preparation of monoclonal antibodies directed toward theimmunogenic proteoliposomes, any technique that provides for theproduction of antibody molecules by continuous cell lines may be used.For example, the hybridoma technique originally developed by Kohler andMilstein (Nature, 256: 495-7,1973), as well as the trioma technique, thehuman B-cell hybridoma technique (Kozbor et al., Immunology Today 4:72),and the EBV-hybridoma technique to produce human monoclonal antibodies,and the like, are within the scope of the present invention. See,generally Larrick et al., U.S. Pat. No. 5,001,065 and references citedtherein. Further, single-chain antibody (SCA) methods are also availableto produce antibodies against polypeptides encoded by a eukaryoticnucleotide sequence of the invention (Ladner et al., U.S. Pat. Nos.4,704,694 and 4,976,778).

[0112] Murine monoclonal antibodies can be “humanized” by knowntechniques.

[0113] The monoclonal antibodies may be human monoclonal antibodies orchimeric human-mouse (or other species) monoclonal antibodies. Thepresent invention provides for antibody molecules as well as fragmentsof such antibody molecules.

[0114] Those of ordinary skill in the art will recognize that a largevariety of possible moieties can be coupled to the resultant antibodiesor preferably to the stabilized trimers or to other molecules of theinvention. See, for example, “Conjugate Vaccines”, Contributions toMicrobiology and Immunology, J. M. Cruse and R. E. Lewis, Jr (eds.),Carger Press, New York, 1989, the entire contents of which areincorporated herein by reference.

[0115] Coupling may be accomplished by any chemical reaction that willbind the two molecules so long as the antibody and the other moietyretain their respective activities. This linkage can include manychemical mechanisms, for instance covalent binding, affinity binding,intercalation, coordinate binding and complexation. The preferredbinding is, however, covalent binding. Covalent binding can be achievedeither by direct condensation of existing side chains or by theincorporation of external bridging molecules. Many bivalent orpolyvalent linking agents are useful in coupling protein molecules, suchas the antibodies of the present invention, to other molecules. Forexample, representative coupling agents can include organic compoundssuch as thioesters, carbodiimides, succinimide esters, disocyanates,glutaraldehydes, diazobenzenes and hexamethylene diamines. This listingis not intended to be exhaustive of the various classes of couplingagents known in the art but, rather, is exemplary of the more commoncoupling agents (see Killen and Lindstrom, J. Immunol. 133:1335-2549,1984; Jansen, F. K, et al., Imm. Rev. 62:185-216, 1982; and Vitetta etal., supra).

[0116] Preferred linkers are described in the literature. See, forexample, Ramakrishnan, S., et al., Cancer Res. 44: 201-208 (1984),describing the use of MBS (M-maleimidobenzoyl-N-hydroxysuccinimideester). See also Umemoto et al., U.S. Pat. No. 5,030,719, describing theuse of a halogenated acetyl hydrazide derivative coupled to an antibodyby way of an oligopeptide linker. Particularly preferred linkersinclude: (i) EDC (1-ethyl-3-(3-dimethylamino-propyl) carbodiimidehydrochloride; (ii) SMPT(4-succinimidyloxycarbonyl-alpha-methyl-alpha-(2-pyridyl-dithio)-toluene(Pierce Chem. Co., Cat. (21558G); (iii) SPDP (succinimidyl-6[3-(2-pyridyldithio) propionamido] hexanoate (Pierce Chem. Co., Cat#21651G); (iv) Sulfo-LC-SPDP (sulfosuccinimidyl 6[3-(2-pyridyldithio)-propianamide] hexanoate (Pierce Chem. Co. Cat.#2165-G); and (v) sulfo-NHS (N-hydroxysulfo-succinimide: Pierce Chem.Co., Cat. #24510) conjugated to EDC.

[0117] The linkers described above contain components that havedifferent attributes, thus leading to conjugates with differingphysio-chemical properties. For example, sulfo-NHS esters of alkylcarboxylates are more stable than sulfo-NHS esters of aromaticcarboxylates. NHS-ester containing linkers are less soluble thansulfo-NHS esters. Further, the linker SMPT contains a stericallyhindered disulfide bond, and can form conjugates with increasedstability. Disulfide linkages, are in general, less stable than otherlinkages because the disulfide linkage is cleaved in vitro, resulting inless conjugate available. Sulfo-NHS, in particular, can enhance thestability of carbodimide couplings. Carbodimide couplings (such as EDC)when used in conjunction with sulfo-NHS, forms esters that are moreresistant to hydrolysis than the carbodimide coupling reaction alone.

[0118] Complexes that form with molecules of the present invention canbe detected by appropriate assays, such as the direct binding assaydiscussed earlier and by other conventional types of immunoassays.

[0119] In a preferred embodiment, one could screen a phage displaylibrary looking to find antibodies to a given protein or find ligandsthat will bind to the protein.

[0120] One can also use these proteoliposomes to screen libraries for adesired compound. One can also use these proteoliposomes to screencomplex chemical libraries of small molecular weight (<1000 daltons)compounds to identify high-affinity ligands. These compounds could serveas lead compounds for the discovery of agonistic and antagonistic drugs.

[0121] If one knows a ligand that interacts with the protein, one canuse these proteoliposomes in assays to screen for compounds thatmodulate such interactions with the protein. For example, in theaforementioned CCR5/CD4-containing proteoliposomes, one can add theoligomeric gp120 or oligomeric gp160 to the mixture and add othercompounds to see their effect on the formation or stability of theCD4/gp120/CCR5 complex.

[0122] One can also use the antibody tag to reverse-orient theproteoliposome. As used herein a reverse-oriented protein will have theportion of the protein that is normally present intracellularly presenton the surface of the proteoliposome. Then one can screen for compoundsor proteins that affect intracellular interactions. For example, one canlook at the binding of intracellular as well as extracellular ligands,as well as compounds or proteins that will affect intracellular as wellas extracellular binding.

[0123] One can also use this method to identify small antagonists in anassay that looks at compounds that affect binding of a known ligand. Forinstance, the entry of human immunodeficiency virus (HIV-1) into hostcells typically requires the sequential interaction of the gp120exterior envelope glycoprotein with the CD4 glycoprotein and a chemokinereceptor on the cell membrane. CD4 binding induces conformationalchanges in gp120 that allow high-affinity binding to the chemolinereceptor. The chemokine receptor CCR5 is the principal HIV-1 coreceptorused during natural infection and transmission. Individuals withhomozygous defects in CCR5 are healthy but relatively resistant to HIV-1infection. Although some HIV-1 isolates can be adapted in tissue cultureto replicate on cells lacking CD4, binding to the chemoline receptorappears to be essential for virus entry into the host cell. Theseobservations suggest that inhibiting the gp120-CCR5 interaction might bea useful therapeutic or prophylactic approach to HIV-1 infection. Achemokine analogue, AOP-RANTES (Simmons, G. et al. (1997), Science: 276:276-279), and a small molecular weight compound (TaKeda) (Baba, M. etal. (1999), Proc. Natl. Acad. Sci. USA 96: 5698-5703) have beenidentified that bind CCR5 and inhibit HIV-1 infection in tissue culture,although clinical utility remains to be demonstrated.

[0124] When solubilized using specific detergent and salt conditions,human CCR5 can retain its ability to bind HIV-1 gp120-CD4 complexes andconformation-dependent monoclonal antibodies (Mirzabekov et al., JBC).However, the detergent-solubilized CCR5 exhibits very stringentrequirements with respect to the conditions under which nativeconformation is retained and has limited longevity. Thus, it isimpractical to use purified preparations of solubilized CCR5 inscreening assays. CCR5-proteoliposomes have homogeneous, native CCR5affixed to the surface of a paramagnetic bead in an oriented manner. Thepreparation of CCR5-proteoliposomes is relatively independent of theCCR5 density on the surface of the cells used as a source of thechemokine receptor, and also allows the concentration of CCR5 on thebead surface. A lipid bilayer, such as that reconstituted around thebead, provides a natural membrane environment for the CCR5 protein,allowing long-term maintenance of the native CCR5 conformation.

[0125] Accordingly, the present method creates an easily manipulablespherical lipid bilayer containing a relatively large amount of pure,oriented and stable integral membrane protein. This permits theseproteins to be used in applications that have previously been restrictedto the use of soluble purified proteins.

[0126] As a specific example, paramagnetic, nonporous beads surroundedby a lipid membrane bilayer containing human gp 120-CCR5 mixtures in anative conformation can be prepared as set forth below. Human CCR5 canbe expressed in Cf2Th canine thymocytes transfected with acodon-optimized CCR5 gene. The CCR5 protein contains a C-terminalnonapeptide (C9) tag that is recognized by the 1D4 monoclonal antibody.In a first approach, lysates from CCRS-expressing Cf2Th cells(Cf2Th-CCR5) were prepared using the detergents shown to allow retentionof native CCR5 formation. CCR5 was affinity-purified from the lysatesusing 1D4Sepharose beads and eluted using the C9 nonapeptidecorresponding to the C-terminal epitope tag.

[0127] Paramagnetic beads were conjugated with both the 1D4 antibody andthe streptavidin. The 1D4 antibody allowed simple purification andconcentration of CCR5 from cell lysates, as well as its orientation onthe bead surface. The streptavidin allowed stable and saturatingmembrane reconstitution around the bead. A 10:1 molar ratio of 1D4antibody:streptavidin was found to be optimal with respect to thehighest density of reconstituted CCR5 and the completeness of themembrane in the paramagnetic proteoliposomes (data not shown). It ispossible to vary the antibody:streptavidin ratio, if necessary, from100:1 to 1:1000 or less in different applications.

[0128] CCR5-proteoliposomes prepared by this approach exhibited the mostefficient recognition by the 2D7 conformation-dependent antibody. Infact, recognition by the 2D7 antibody was practically equal to that ofthe 5C7 antibody (data not shown), indicating that the vast majority ofCCR5 in these proteoliposomes preparations is in a native conformation.The envelope glycoprotein can be combined on the proteoliposome asdiscussed above.

[0129] The paramagnetic proteoliposomes are stable for extended periodsof time. The integrity of the conformational dependent epitope on theproteins such as the gp120 proteins is maintained for extended periodsof time permitting the uses described above.

EXAMPLES Example 1

[0130] Conformational Characterization of Proteoliposomes Containinggp160

[0131] Envelope Glycoprotein Constructs

[0132] The envelope glycoprotein constructs were derived from theprimary R5 HIV-1 isolates YU2 and JR-FL and the X4, TCLA-adapted isolateHXBc2. The coding sequences for the YU2 envelope glycoprotein wereobtained from the pSVIIIenvYU2 expression plasmid. The JR-FL envelopeglycoprotein coding sequence, which contains a CD5 heterologous leadersequence m place of the normal JR-FL leader, was obtained from the AIDSrepository and subcloned into the pcDNA 3.1(−) (Invitrogen) expressionplasmid. The HXBc2 construct was codon-optimzed for mammalian expressionusing overlapping primers and PCR and subcloned into pcDNA3.1(+)(Invitrogen). A sequence coding for the heterologous CD5 signal sequencewas subcloned to replace the endogenous HXBc2 leader sequence. To allconstructs, the cytoplasmic tail truncation was generated byintroduction of a stop codon in place of the codon for amino acid 712and the sequence coding for the C9-tag TETSQVAPA was added according tothe QuikChange protocol immediately before the stop codon. To createcovalently linked gp120-gp41 glycoproteins, the proteolytic cleavagesite between gp120 and gp41 was disrupted by replacing the arginines 508and 511 with serines by QuikChange site-directed mutagensis. Thesemodifications resulted in constructs encoding cleavage-deficientgp160ΔCT envelope glycoproteins used to generate the proteoliposomes.Amino acid residue numbers are designated according to the prototypicHXBc2 sequence. The constructs were sequenced and introduction of thedesired mutations was confirmed by this method

[0133] Envelope Glycoprotein Expression

[0134] Plasmids expressing the gp160ΔCT glycoproteins (2 μg DNA per 100mm dish of cells) were cotransfected into 293T cells with (YU2) orwithout (JR-FL, HXBc2) the HIV-1 Tat expressor plasmid pSVTat (0.5 μg),using Effectene reagent (QIAGEN) following the manufacturer's protocol.Thirty-six hours after transfection, cells expressing the envelopeglycoproteins were harvested with PBS containing 5 mM EDTA.

[0135] Coating of Dynabeads M-450

[0136] Tosyl-activated Dynabeads (Dynal, Inc., Lake Success, N.Y.) wereconjugated with the 1D4 antibody (National Cell Culture Center,Minneapolis, Minn.) according to the manufacture's protocol. The 1D4murine antibody recognizes the C9 epitope tag and was used to capturethe envelope glycoproteins on the Dynal beads.

[0137] Preparation of Lipid Solutions for Membrane Reconstitution

[0138] Lipids were obtained as chloroform solutions from Avanti PolarLipids (Alabaster, Ala.). The following lipids were used:1-Palmitoyl-2-Oleoyl-sn-Glycero-3-Phosphocholine (POPC),1-Palmitoyl-2-Oleoyl-sn-Glycero-3-Phosphoethanolamine (POPE) andDimyristoylphosphatidic acid (DMPA) and Cholesterol at molarconcentrations 45:25:20:10. The lipid mixture was dried in a 2-mlpolyethylene tube under a vacuum until all of the solvent was removed.PBS was added to the tube and a liposomal solution was obtained byultrasonication for 5 minutes in an ice bath using an UltrasonicProcessor (Heat Systems, Inc., Farmingdale, N.Y.). Liposomal solutionsof the head group-modified synthetic lipids1,2-dioleyl-sn-glycero-3-phosphoethanolamine-n-(biotinyl)(Biotinyl-DOPE) and dioleoylphosphoethanolamine-lissamine rhodamine B(Rho-DOPE), at a final concentration of 1 mg/ml, were preparedseparately using the same protocol.

[0139] Formation of Proteoliposomes

[0140] For the preparation of 4×10⁸ proteoliposomes, approximately 2×10⁷gp160ΔCT-expressing 293T cells were lysed in 5 ml solubilization buffer(100 mM (NH₄)₂SO₄, 20 mM Tris-HCI (pH 7.5), 1% (w/v) Cymal™-5 andProtease Inhibitor Mixture (one tablet of Complete™[Boehringer Mannheim]per 50 ml) at 4° C. for 30 minutes on a rocking platform Cell debris waspelleted by centrifugation for 30 minutes at 13,000 g. The clearedlysate was incubated with 4×10⁸ 1D4-conjugated Dynal beads for 16 hoursat 4° C. on a rocking platform. After recovery of the beads, they wereextensively washed in solubilization buffer. For the formation of thelipid membrane, beads coated with gp160ΔCT glycoprotein were incubatedfor 15 minutes at RT with 1 ml solubilization buffer containing 2 mg oflipid mixture, and, if fluorescence labeling or biotinylation wasdesired, with 1% of Rho-DOPE or biotin-PE. The detergent was then slowlyremoved by dialysis for 24 hours at 4° C. against PBS, using a 10 kDamolecular weight cutoff dialysis membrane (Slide-A-Lyzer® 10 K [Pierce,Rockford, Ill.]). The excess of unbound lipid and residual detergent wasremoved on a magnetic separator in one washing step with PBS.Proteoliposomes were stored in PBS with 0.1% BSA and 0.1% Na₂N₃ at 4° C.for up to three months.

[0141] Analysis of Protein Composition of the Proteoliposomes

[0142] Approximately 5×10⁷ PLs and control beads were incubated at 100°C. in reducing SDS-sample buffer for 5 minutes, separated on a 7.5%SDS-polyacrylamide gel and stained with Coomassie blue. For the analysisof fractions eluted by molecular exclusion, Western blotting wasperformed under either non-reducing or reducing conditions. To determinethat gp120 was present in the eluted protein peaks, samples from eachpeak were incubated for 5 minutes at 100° C. in sample buffer containing2% BME and separated on a 7.5% SDS-polyacrylamide gel (FIG. 2B). Toconfirm that oligomeric forms of envelope glycoproteins could bedetected in the high molecular weight peaks, the fractions were dilutedin sample buffer lacking BME and separated on a 3-8% SDS-polyacrylamidegel. A sample from the peak consistent with a trimer (fraction 3, FIG.4) was also analyzed in the presence of BME. Subsequently, proteins wereelectrophoretically transferred onto a 0.45 μm Hybond-C extra membrane(Amersham). The gp160ΔCT glycoproteins present in each column fractionwere then detected by anti-gp120 rabbit serum and anti-rabbitIgG-horseradish peroxidase (HRP) (Sigma).

[0143] Molecular Exclusion Chromatography

[0144] The gp160ΔCT glycoproteins captured onto Dynal beads were elutedfrom the beads for molecular exclusion chromatography undernon-denaturing conditions as follows. The beads were incubated in 0.5 MMgCl₂, 1% CHAPS, and 0.2 mM C9 peptide (eptide sequence: TETSQVAPA) at37° C. for 30 minutes. Approximately 5 μg of eluted gp160ΔCTglycoproteins were loaded onto a Superdex 200 column (Amersham PharmaciaBiotech) in a 200 μl volume. The column was then eluted with PBScontaining 1% CHAPS at a rate of 0.5 ml/min for 40 minutes. The elutedprotein was detected by measuring the optical density at 280 nm (OD280)using a Varian ProStar System (Varian Analytical Instruments). Fractionsof the flow-through were collected at 2 minute intervals using a VarianDynamax Fraction Collector. The fractions were further analyzed byreducing and non-reducing SDS-PAGE and Western blotting using apolyclonal anti-gp120 rabbit serum for detection of HIV-1 envelopeglycoproteins (FIGS. 4B-C). A mixture of high molecular weight proteinmarkers (Amersham Pharmacia Biotech) was eluted under identicalconditions to calibrate the column. Flow cytometric analysis of gp160ΔCTon proteoliposomes and 293T cells For the comparison of antibody bindingto either cleavage-defective or cleavage-competent gp160ΔCTglycoproteins, 293T cells were transfected with plasmids expressing thetwo envelope glycoprotein variants. Approximately 10⁶ cells per samplewere harvested with PBS containing 5 mM EDTA and washed once in FACSbuffer (PBS, 2% FCS, 0.02% Na₂N₃). The cells were incubated for 1 hourat RT with the indicated amounts of antibodies in a volume of 100 μl.After two washing steps in FACS buffer, the cells were incubated for 30min with a R-Phycoerythin (PE)-conjugated F(ab′)2 goat anti-humanantibody (Jackson Immuno Research Laboratories, Inc., West Grove, Pa.),washed twice and analyzed with a FACScan flow cytometer and CellQuestsoftware (Beckton Dickinson, San Jose, Calif.). For FACS analysis ofPLs, staining was performed as described above. Staining for membraneintegrity of the PLs was performed using a PE-conjugated goat anti-mouseIgG (Boehringer Mannheim, Indianapolis, Ind.) or Avidin-FIFC (Sigma).The following ligands were used for staining of the envelopeglycoproteins: soluble CD4, the potently neutralizing CD4BS antibodyIgG1b12 (kindly provided by Dennis Burton), the F105 CD4BS antibodykindly provided by Marshal Posner), the strain-restricted neutralizingV3 loop antibody 39F, the non-neutralizing C1/C4 antibody A32, thenon-neutralizing C1/C5 antibody C11, the CD4-induced 17b antibody (allkindly provided by James Robinson) and the broadly neutralizing gp41antibody 2F5 (kindly provided by Hermann Katinger).

[0145] Creation of gp160ΔCT Proteoliposonmes

[0146] Paramagnetic proteoliposomes (PLs) containing the HIV-1 envelopeglycoprotein were created according to methods established forsolubilization and membrane reconstitution of the CCR5 chemokinereceptor (Mirzabekov et al., Nat. Biotechnol. 18:649-654 (2000)).Briefly, cells transiently expressing the HIV-1 YU2, JR-FL, or HXBc2gp160ΔCT glycoproteins, which contain an alteration of the gp120-gp41cleavage site and deletion of the gp41 cytoplasmic tail, were lysed inbuffer containing Cymal-5 detergent. The gp160ΔCT glycoproteins werethen captured onto Dynal beads conjugated to the 1D4 antibody, whichrecognizes a C9 peptide tag affixed to the gp160ΔCT C-terminus.Following addition of membrane lipids and dialysis of the Cymal-5detergent, an artificial lipid bilayer is formed around the beadsurface. Thus, pure, properly oriented HIV-1 envelope glycoproteins,embedded in a natural membrane environment were incorporated into aneasily manipulable solid support. A schematic of the gp160proteoliposomes is shown in FIG. 1.

[0147] Analysis of Proteoliposome (PL) Protein Composition

[0148] The gp160ΔCT PLs were boiled in sample buffer and analyzed byreducing SDS-PAGE to determine their protein composition. As a positivecontrol, cell lysates containing transiently expressed gp160ΔCTglycoproteins were precipitated with the F105 anti-gp120 antibody andprotein A-Sepharose and analysed in parallel. PLs lacking the gp160ΔCTglycoproteins were treated similarly and served as a negative control.As seen in FIG. 2A, a band migrating at a position similar to thegp160ΔCT glycoproteins positive control band was observed among theproteins released from the gp160ΔCT PLs. No such band was observed inthis molecular weight range in the gp160ΔCT glycoprotein-deficient PLcontrol sample. Apart from a 50 kD band corresponding to the 1D4antibody heavy chain, a light chain band not retained on the gel, andthe band, only minor impurities were detected in the gp160ΔCT PLs. Thetotal amount of gp160ΔCT glycoproteins captured onto 5×10⁷proteoliposomes was estimated to be 1-2 μg, as determined by thepurified recombinant gp160ΔCT glycoproteins control of knownconcentration.

[0149] To examine the exposure of the HIV-1 envelope glycoproteins onthe proteoliposome surface, the gp160ΔCT PLs were stained with theIgG1b12 anti-gp120 antibody and a mixture of sera from HIV-1-infectedindividuals and analyzed by fluorescent activated cell sorting (FACS).The IgG1b12 antibody recognizes a conformation-dependant gp120 epitopenear the CD4 binding site. The forward scatter versus sideward scatterplot showed mostly single PLs. Doubles and other multiples of PLs weregenerally observed to be less than 20% of the total events (data notshown). A gate was created to analyze only single PLs and was used forall further FACS analyses. The narrow distribution of the fluorescenceintensity associated with each FACS peak following antibody stainingsuggests that the gp160ΔCT PLs have nearly uniform protein content (FIG.2B).

[0150] Characterization of the Size of the gp160ΔCT PL EnvelopeGlycoproteins

[0151] The gp 160ΔCT glycoproteins were eluted from the reconstitutedPLs under non-denaturing conditions by incubation with 0.2 mM C9 peptidein the presence of 1% CHAPS detergent and 0.5 M NaCl. The elutedenvelope glycoproteins were analyzed by size-exclusion chromatography ona Superdex 200 column equilibrated in PBS/1% CHAPS buffer. Thechromatogram for the HIV-1 JR-FL gp160ΔCT envelope glycoproteins isshown in FIG. 4A. Parallel studies using the YU2 gp160ΔCT glycoproteinsyielded a similar profile (data not shown). The fractions of theresolved JR-FL glycoproteins were collected and analyzed by SDS-PAGE andWestern blotting (FIG. 4B).

[0152] The column was calibrated with molecular weight standards,allowing the apparent molecular size of the major peak to beapproximated as 580 kD. As the gp120 glycoprotein monomer was resolvedby size-exclusion chromatography with an apparent molecular weight of180 kD (data not shown and Yang et al., J. Virol. 74:5716-5725 (2000)),a mass of 580 kD is consistent with that of a trimeric gp160ΔCT envelopeglycoprotein complex. The fastest migrating protein peak was detectedjust after the void volume and apparently consists of gp160ΔCTglycoprotein aggregates as determined by its migration pattern in thecolumn and by the Western blot results (FIG. 4). This aggregate peak haspreviously been observed in molecular exclusion chromatography ofsoluble GCN4stablized gp140 trimers (Yang et al., J. Virol. 74:5716-5725(2000)). Most of the gp160ΔCT glycoprotein eluted in fractions 3 and 4,with fraction 3 corresponding to the mass of 580 kD. When all of the gelfiltration fractions were subjected to non-reducing SDS-PAGE followed byWestern blotting, fractions 3 and 4, corresponding to trimeric envelopeglycoproteins, were found to separate into trimers, dimers and monomers.The greatest degree of gel-stable trimeric glycoproteins were detectedin fraction 3 (FIG. 4C). Most of the glycoproteins migrating withmolecular weight corresponding to trimers in this fraction could benearly totally reduced to a monomeric gp160ΔCT band by treatment with 2%β-mercaptoethanol and boiling, although bands migrating in mannerconsistent with trimers and dimers could still be observed (FIG. 4C).

[0153] Characterization of the Proteoliposome Membrane

[0154] The formation of the lipid membrane was examined by FACS analysisand fluorescent microscopy. According to our model, the murine 1D4capture antibody would be expected to be partially occluded by areconstituted lipid membrane (see FIG. 1). Thereby, binding ofanti-mouse IgG antibody should be impaired on proteoliposomes whencompared to beads without a membrane. A more than 3-fold decrease ofanti-mouse-PE signal could be observed on PLs containing a reconstitutedlipid membrane versus beads without lipid reconstitution (FIG. 5B, peak1 compared to peak 2), indicating the presence of at least a partiallipid membrane.

[0155] The presence of the lipid membrane was also examined by FACSanalysis after reconstitution of the membrane with biotinylated lipid at1% (w/w) of total lipids. The PLs were stained with avidin-FITC andshowed more than a 20-fold higher signal on fully reconstituted PLscompared to gp160ΔCT glycoprotein-containing beads without lipidreconstitution (FIG. 5B). We then confirmed the presence of a lipidbilayer by visualizing the incorporation of rhodamine-conjugated lipid(rhodamine-DOPE) into the PL membrane by fluorescent microscopy (FIG.3). Bright fluorescence could be observed in the rhodamine-labelled PLs(FIG. 3B) compared to negligible background fluorescent emission withuntreated beads (FIG. 3A).

[0156] 2F5 Antibody Binding to gp160ΔCTPLs

[0157] The epitope of the neutralizing antibody 2F5, ELDKWAS, issituated proximal to the viral membrane in the gp41 ectodomain. Todetermine the influence of the reconstituted membrane on this importantneutralizing determinant, we used the 2F5 antibody to probe PLsreconstituted with a membrane and PLs coated with gp160ΔCT glycoproteinbut devoid of a membrane. Since sequence variation in the HIV-1 YU2strain alters 2F5 antibody recognition, PLs with gp160ΔCT glycoproteinsderived from the primary isolate JR-FL and the T-Cell line adaptedisolate HXBc2 were examined. The 2F5 antibody bound to HXBc2 gp160ΔCTglycoprotein PLs with a reconstituted membrane with roughly a tenfoldhigher affinity than it did to HXBc2 gp160ΔCT glycoprotein on beadswithout a membrane. The same could be observed with JR-FL PLs, althoughthe effect was less pronounced (FIG. 6). To rule out unspecific antibodybinding effects caused by the presence of the membrane, binding of thegp120-specific F105 antibody to PLs with and without a membrane wasexamined. No affinity difference could be detected using the F105antibody (data not shown).

[0158] Antigenic Characterization of the gp160ΔCTPLs

[0159] To confirm the native conformation of the gp160ΔCT glycoproteinson the surface of the PLs, we compared the binding of a panel ofconformationally-sensitive ligands to gp160ΔCT glycoproteins on PLs withbinding to gp160ΔCT glycoproteins expressed on the surface oftransiently-expressing cells. The antibodies were selected to probeseveral faces of the HIV-1 envelope glycoproteins including theneutralization-relevant epitopes overlapping the CD4 binding site(CD4BS). We used the potently neutralizing CD4BS antibody IgG1b12, theless potent neutralizing CD4BS-antibody F105, and soluble CD4 (sCD4) toconfirm the correct native conformation of the CD4BS. In addition, weprobed gp160ΔCT PLs with the strain-restricted neutralizing V3-loopantibody 39F, and the non-neutralizing antibodies A32 and C11. Thebinding characteristics of all antibodies to the gp160ΔCT PLs comparedto gp160ΔCT glycoproteins expressed on the cell surface were virtuallyindistinguishable (FIG. 7). The only difference in binding profiles wasobserved for soluble CD4 (sCD4). For the PLs, sCD4 displayed an almosttenfold higher affinity than it did for gp160ΔCT glycoproteins expressedon cell surfaces. This affinity difference may be a consequence ofbetter exposure of gp160ΔCT glycoproteins on the PLs due to the lack ofthe cellular glycocalix and cellular protein components. The observedaffinities calculated for the antibodies were in the low nanomolarrange, consistent with previously reported values (Posner, et al., J.Acquir. Immune Defic. Syndr. 6:7-14 (1993); Roben, et al., J. Virol.68:4821-4828 (1994)). For example, the affinity of the antibody IgGb12,as determined by the concentration necessary to achieve half-maximalbinding, was calculated to be 6 nM. The affinity of the non-neutralizingC11 antibody was at least 10-fold lower than that of the neutralizingIgG1b12 antibody. This underestimates the affinity difference betweenthe two antibodies because saturation binding was not achieved with theC11 antibody.

[0160] CD4 Induction of the 17b Epitope

[0161] One functional conformational change characteristic of nativegp120 is the induction of the 17b antibody epitope by CD4 (Sullivan etal., J. Virol. 72:4694-4703 (1998)). To test whether the gp160ΔCTglycoproteins on PLs exhibit this property of gp120 expressed on thecell surface, gp160ΔCT glycoprotein PLs and 293T cells expressingcleavage-competent envelope glycoproteins were preincubated with sCD4prior to binding of the 17b antibody. 17b binding was detected bystaining with a PE-conjugated anti-human IgG antibody followed by FACSanalysis. The binding of the 17b antibody was increased by 63% afterpreincubation with sCD4 on cleavage competent gp160ΔCT glycoproteinexpressed on cells compared to a 46% increase on gp160ΔCT PLs (FIG. 8).

[0162] Characterization of Cleavage-Deficient gp160ΔCT Glycoproteins

[0163] Deletion of the cleavage site for the host protease furin resultsin the expression of uncleaved gp160ΔCT glycoprotein precursor proteinson the surface of transfected cells. This modification abrogates thedissociation of gp120 from the cell surface and therefore increases theamount of envelope glycoproteins retained on the cell surface orcaptured on the surface of the PLs. In addition, a cytoplasmic taildeletion was introduced into the gp160 glycoprotein in order to increasecell surface expression (ACT). Cell surface expression levels ofcytoplasmic tail-deleted envelope glycoproteins increased 8-fold overfull-length constructs containing the intact gp160 cytoplasmic tail(data not shown).

[0164] It is possible that the deletion of the cleavage site mightdistort envelope glycoprotein conformation and result in a conformationnot representative of functional, cleaved envelope glycoprotein. Toassess the effects of the cleavage site deletion, we analyzed thebinding properties of a panel of conformationally-sensitive ligands tocleavage-defective and cleavage-competent gp160ΔCT glycoproteinsexpressed on the cell surface. FACS analysis was performed by stainingthe gp160ΔCT expressed on 293T cells with increasing concentrations ofligands. No significant affinity difference was observed betweencleavage-competent gp160ΔCT glycoproteins and cleavage-defectiveenvelope glycoproteins for any of the ligands tested (FIG. 9). However,one notable difference in the binding profiles was observed. The bindingprofile of sCD4 on cells expressing cleavage-competent gp160ΔCTglycoproteins had a biphasic shape. As sCD4 is known to induce sheddingof gp120 from envelope glycoprotein complexes (Moore, et al., Science250:1139-1142 (1990)), CD4 binding to the cleavage-competent gp160ΔCTglycoprotein may induce shedding or other conformational changes thatresult in a biphasic binding profile.

[0165] To confirm the processing of the cleavage-competent envelopeglycoproteins, 293T cells were transfected with cleavage-competent and-defective envelope glycoprotein constructs. Proteins expressed on thecell surface were iodinated by lactoperoxidase. After detergent lysisand precipitation with the F105 antibody, iodinated proteins wereanalyzed by SDS-PAGE. The ratio of unprocessed gp160ΔCT precursorproteins to cleaved gp120 was determined to be about 1:1 (data notshown).

Example 2

[0166] Proteoliposomes Containing CCR5

[0167] Construction and Expression of Codon-Optimized CCR5 (synCCR5)

[0168] The analysis of codon usage for 45 GPCRs representing differentprotein subfamilies was performed with GenBank™ data and softwaredeveloped by the University of Wisconsin Genome Sequence Group. Thesequence encoding human CCR5 was optimized for mammalian cell codonusage (Andre, S., et al. (1999). J. Virology 72:1497-1503) utlizing thefollowing codons: alanine (GCC), arginine (CGC), asparagine (AAC),aspartic acid (GAC), cysteine (TGC), glutamic acid (GAG), glutamine(CAG), glycine (GGC), histidine (CAC), isoleucine (ATC), leucine (CTG),lysine (AAG), methionine (ATG), phenylalanine (TTC), proline (CCC),serine (TCC), threonine (ACC), tryptophan (TGG), tyrosine (TAC), andvaline (GTG). The 5′ and 3′ sequences flanking the CCR5 coding sequencewere modified. Following restriction sites for EcoRV, EcoRI and HindIII,the Kozak consensus (GCCGCCACCATGG) (SEQ ID NO:1) was placed immediately5′ to the CCR5 reading frame. A sequence encoding a single glycineresidue followed by the bovine rhodopsin C9 peptide tag (TETSQVAPA) (SEQID NO:2) was introduced immediately 5′ to the natural stop codon ofCCR5. At the 3′ end of the epitope-tagged CCR5 gene, XbaI, SalI, andNotI restriction sites were introduced. Analogous constructs were madefor the wild-type human CCR5 gene and the bovine rhodopsin gene, exceptthat the codons were not altered and, in the latter case, the C-terminalC9 sequence was naturally present.

[0169] A total of 34 oligonucleotides, each approximately 70 nucleotidesin length, corresponding to the complete sense and antisense strands ofthe synCCR5 gene and flanking sequences, were constructed so thatapproximately 50% of their sequences were complementary to those of eachof the two complementary oligonucleotides from the opposite strand.Oligonucleotides were deprotected in pure ammonium hydroxide at 65° C.for 4 h, after which the ammonium hydroxide was evaporated, and theoligonucleotides were dissolved in water at a final concentration of 2nM. For gene synthesis, the 34 oligonucleotides were separated into fivegroups (6 or 8 oligonucleotides per group) and 25 cycles of polymerasechain reaction were performed using Pfu polymerase (Stratagene, LaJolla, Calif.) and a 3-fold molar excess of the 5′ and 3′ terminaloligonucleotides in each group. This step generated five small segmentsof the synCCR5 gene with complementary and overlapping ends. Equalamounts of each polymerase chain reaction product were combined with a3-fold molar excess of the 5′ and 3′ terminal oligonucleotides of thecomplete synCCR5 sequence. A second round of 25 cycles of polymerasechain reaction yielded the complete synCCR5 sequence. The product wassequenced to ensure that the sequence was correct.

[0170] The synCCR5, wild-type CCR5, and bovine rhodopsin sequences werecloned into the following vectors: PMT4 (a gift from Dr. Reeves,Massachusetts Institute of Technology), PACH (a gift from Dr. Velan,Israel Institute for Biological Research), pcDNA 3.1(+) andpcDNA4/HisMax (Invitrogen), and PND (a gift from Dr. Rhodes, Universityof California, Davis). After cloning of the synCCR5 gene into thepcDNA4/HisMax vector, the sequence encoding the N-terminal HisMax regionwas removed by QuikChange mutagenesis (Stratagene). Different cell lineswere transfected with the synCCR5 and wild-type CCR5 genes using theGenePorter transfection reagent (San Diego, Calif.). Followingtransfection, cells expressing CCR5 were selected with 0.8 mg/ml ofneomycin (G418). Cells expressing the highest surface levels of CCR5were selected by FACS after staining cells with theR-phycoeryrin-conjugated anti-CCR5 antibody 2D7-PE (Pharmingen, SanDiego, Calif.). Among all tested cells (canine thymocytes Cf2Th, humanembryonic kidney cells HEK-293T, COS-1, and HeLa (American Type CultureCollection)), the highest CCR5 expression levels were observed in Cf2Thand HEK-293T cells transfected with synCCR5 gene in the PACH vector. Thehighest synCCR5-expressing clones were selected by FACS from a total of76 clones of Cf2Th cells and 62 clones of HEK-293T cells.

[0171] Radiolabeling and Immunoprecipitation of CCR5

[0172] Approximately 4×10⁶ CCR5-expressing Cf2Th or HEK-293T cells grownto full confluency in 100-mm dishes were washed twice in PBS and starvedfor 1 h at 37° C. in Dulbecco's modified Eagle's medium without cysteineand methionine (Sigma) or in sulfate-free media (ICN, Costa Mesa,Calif.). The starvation medium was removed and 200 TCi each of[³⁵S]methionine and [³⁵S]cysteine or 500 TCi of [³⁵S]sulfate (NEN LifeScience Products) in 4 ml of medium was added to the cells for varioustimes for pulse-chase experiments or overnight (12 h) in all othercases. Cells were washed twice with PBS and lysed in 1 ml ofsolubilization medium composed of 100 mM (NH₄)₂SO₄, 20 mM Tris-HCl (pH7.5), 10% glycerol, 1% (w/v) detergent (see below), and ProteaseInhibitor Mixture (one tablet of Complete™ (Roche MolecularBiochemicals) per 25 ml). The lysate was incubated at 4° C. for 30 minon a rocking platform, and cell debris was removed by centrifugation at14,000×g for 30 min. CCR5 was precipitated with 20 Il of 1D4-Sepharosebeads (Reeves, P., Thurmond, R. L., and Khorana, G. G. (1996) Proc.Natl. Acad. Sci. USA 4: 7784-90) overnight, after which the beads werewashed six times in the solubilization medium and pelleted. An equalvolume of 2×SDS-sample buffer was added to the beads, followed byresuspension and incubation for 1 h at 55° C. Samples were run on 11%SDS-polyacrylamide minigels, which were visualized by autoradiography oranalyzed on a Molecular Dynamics PhosphorImager SI (Sunnyvale, Calif.).

[0173] A total of 18 detergents were tested in the solubilizationbuffers. The detergents, with abbreviations and critical micelleconcentrations in parentheses, were n-octyl-β-D-glucopyranoside (23.4mM), n-decyl-β-D-maltoside (1.8 mM), n-dodecyl-β-D-maltoside (DDM) (0.17mM), cyclohexyl-butyl-β-D-maltoside (Cymal™-4, 7.6 mM),cyclohexyl-pentyl-β-D-maltoside (Cymal™-5, 2.4 mM),cyclohexyl-hexyl-β-D-maltoside (Cymal™-6, 0.56 mM),cyclohexyl-heptyl-β-D-maltoside (Cymal™-7, 0.19 mM),cyclo-hexylpropanoyl-N-hydroxyethylglucamide (108 mM),cyclohexylbutanoyl-N-hydroxyethylglucamide (35 mM),cyclohexylpentanoyl-N-hydroxyethyglucamide (11.5 mM),N-octylphosphocholine (Fos-Choline™8, 114 mM), N-decylphosphocholine(Fos-Choline™10, 11 mM), N-dodecylphosphocholine (Fos-Choline™12, 1.5mM), N-tetradecylphosphocholine (Fos-Choline™14, 0.12 mM), Triton X-100(0.02 mM), CHAPS (8 mM), Nonidet P-40 (0.02 mM), anddiheptanoyl-phosphocholine (DHPC) (1.4 mM). All detergents werepurchased from Anatrace (Maumee, Ohio) except DBPC, which was purchasedfrom Avanti Polar Lipids (Alabaster, Ala.).

[0174] Purification of CCR5

[0175] Stable Cf2Th/PACH/synCCR5 cells grown to full confluency in a150-mm dish were incubated with medium containing 4 mM sodium butyratefor 40 h, washed in PBS, detached by treatment with 5 mM EDTA/PBS,pelleted, and again washed in PBS. Cells were solubilized for 30 minwith 3 ml of the solubilization medium containing Cymal™-5 andcentrifuged for 30 min at 14,000×g. The cell lysate was incubated with50 Il of 1D4-Sepharose beads on a rocking platform at 4° C. for 10-12 h.The Sepharose beads were washed five times with the washing buffer (100mM (NH₄)₂SO₄, 20 mM Tris-HCl (pH 7.5), 10% glycerol, and 1% Cymal™-5)and once with washing buffer plus 500 mM MgCl₂. CCR5 was eluted from thebeads by three successive washes with 50 Il of medium containing 200 IMC9 peptide (SEQ ID NO:2) (TETSQVAPA), 500 mM MgCl₂ 100 mM (NH₄)₂SO₄, 20mM Tris-HCl (pH 7.5), 10% glycerol, and 0.5% Cymal™-5. The totalquantity of harvested CCR5 was estimated by Coomassie Blue staining ofan SDS-polyacrylamide gel run with standard quantities of bovine serumalbumin.

[0176] Binding of HIV-1 gp120 Envelope Glycoproteins to Solubilized CCR5

[0177] Approximately 4×10⁶ Cf2Th/PACH/synCCR5 cells were labeled for 12h with [³⁵S]Met/Cys and lysed in solubilization buffer containing 1%Cymal™-5. One ml of cleared cell lysate was incubated with 100-500 Il ofthe gp120-containing solutions. The unlabeled JR-FL gp120 was producedin Drosophila cells (Wu, L., et al. (1996), Nature 384,179-183), and theADA and 190/197 R/S gp120 glycoproteins were produced from transientlytransfected 293T cells that had been radiolabeled with [³⁵S]Met/Cysovernight. Except in the case of the CD4-independent gp120 variant,190/197 R/S, the gp120 glycoproteins (2-4 lg) were preincubated withsCD4 (2-4 lg) in 20 ml of PBS for 1 h at 22° C. prior to addition to theCCR5-containing lysates. After 12 h at 4° C., the gp120-CCR5 complexeswere precipitated with either the C11 anti-gp120 antibody (kindlyprovided by Dr. James Robinson, Tulane University Medical School) orwith the 1D4 antibody.

[0178] Expression of CCR5 in Mammalian Cells

[0179] We compared the codon usage for opsins, the only GPCRs that arenaturally highly expressed, with the codon usage for 45 other GPCRsrepresenting a spectrum of different GPCR subfamilies. Opsin codons arebiased toward those shown to be optimal for efficient translation inmammalian cells (Andre, S., et al. (1998), J. Virol. 72: 1497-1503),whereas other GPCRs, including CCR5, are associated with codons that aremore random and, in many cases, inefficiently translated (data notshown). A codon-optimized CCR5 gene was designed, synthesized using thepolymerase chain reaction, and transiently expressed in severaldifferent cell lines, using five different expression vectors (pcDNA3.1, PACH, PND, PMT 4, and pcDNA4/HisMax). The level of CCR5 expressiondirected by the codon-optimized gene was 2-5 times that directed by thewild-type CCR5 gene. Among the cell lines tested, CCR5 expression wasthe highest in Cf2Th canine thymocytes (data not shown), so these cellswere used to generate stable cell lines. The PACH vector was used toexpress the codon-optimized gene encoding human CCR5 containing a9-residue C-terminal epitope tag (the C9 tag) derived from bovinerhodopsin. The presence of the C9 tag allows recognition of the CCR5protein by the 1D4 antibody (Oprian, D. D., et al. (1987), Proc. Natl.Acad. Sci. U.S.A. 84: 8874-8878). CCR5 expression in the stable cellline, designated Cf2Th/PACH/synCCR5, could be enhanced 2-3 fold bytreatment of the cells with sodium butyrate. Following this treatment,approximately 3-5 Ig of CCR5 of high purity could be isolated from 10⁷Cf2Th/PACH/synCCR5 cells, using techniques described below.

[0180] Precursor and Mature Forms of CCR5

[0181] CCR5 synthesis and turnover in Cf2Th cells were studied bypulse-chase analysis. A precursor of approximately 40 kDa chased intothe mature form of CCR5, which migrated as a wide band of approximately43 kDa The CCR5 precursor exhibited a half-life of approximately 25 min.The half-life of the mature form of CCR5 was 11-14 h, regardless ofwhether CCR5 expression was directed by the wild-type or codon-optimizedCCR5 gene. The half-lives of the precursor and mature forms of CCR5 inHEK-293 cells were similar to those in Cf2Th cells (data not shown). Inseveral different cell lines, a lower molecular mass (approximately 36kDa) form of CCR5 appeared in parallel with the mature protein. Thislower molecular mass form of CCR5 was expressed at lower levels than themature form of CCR5 and has not been completely characterized. Itsidentity as a CCR5 isoform was confirmed by its precipitation by the 1D4antibody and the anti-CCR5 antibody 2D7 and by mass spectrometry.

[0182] Solubilization of Native CCR5

[0183] Membrane protein purification requires solubilization of themembranes, typically through the use of detergents. A broad spectrum ofconditions was studied to arrive at the composition of the buffer thatallowed solubilization and isolation of native CCR5. This optimizationwas guided by a comparison of the amount of solubilized CCR5 capable ofbeing precipitated by the 2D7 antibody, which recognizes aconformation-dependent CCR5 epitope (Wu, L., et al. (1997), J. Exp. Med.186: 1373-1381), with that able to be precipitated by the 1D4 antibodydirected against the linear C9 epitope tag. In this manner, thepercentage of solubilized CCR5 remaining in a native conformation couldbe estimated. Eighteen detergents, most of which were designedspecifically for the extraction and purification of membrane proteins,were studied. In terms of the quantity of isolated CCR5 protein, as wellas the percentage of protein in a conformation able to be recognized bythe 2D7 antibody, the most effective detergents were DDM, Cymal™-5, andCymal™-6. Of these detergents, Cymal™-5 exhibits the highest criticalmicelle concentration (2.4 mM), facilitating dialysis of the detergentfrom the protein solution for the purposes of membrane reconstitutionand/or crystallization. We also found that a CCR5 conformation competentfor binding HIV-1 gp120 was best preserved in buffers containingCymal™-5 (see below). Therefore, Cymal™-5 was used for furtherrefinement of the CCR5 solubilization/isolation protocol, examining anumber of variables (salt composition and concentration, pH,temperature, and minor additives) known to influence the stability ofsolubilized proteins (Hamaguchi, K (1992) The Protein Molecule.Conformation, Stability and Folding, Japan Scientific Societies Press,Springer-Verlag, New York). Ammonium sulfate and glycerol were found toprolong the existence of a CCR5 conformation capable of being recognizedby the 2D7 antibody (data not shown). The optimized CCR5 solubilizationbuffer was composed of 100 mM (NH₄)₂SO₄, 20 mM Tris-HCl (pH 7.5), 10%glycerol, and 1% Cymal™-5.

[0184] CCR5-Expressing Cells.

[0185] The cell line (Cf2Th/CCR5) stably expressing approximately 10⁶molecules of CCR5 per cell was generated by transfection of Cf2Th caninethymocytes with the above-described codon-optimized CCR5 gene. TheC-terminus of the expressed CCR5 consists of a glycine residue followedby the C9 nonapeptide TETSQVAPA (SEQ ID NO: 2), which contains theepitope for the 1D4 antibody. Wild-type and C-terminally tagged CCR5molecules have been shown to be functionally comparable. Cf2Th/CCR5cells grown to full confluency in 150 mm dishes were harvested using 5mM EDTA in PBS, washed in PBS, pelleted and frozen until needed.

[0186] Radiolabeling of Cells Expressing CCR5 or gp120.

[0187] Cf2Th/CCR5 cells were radiolabeled in 150 mm dishes for 12 hourswith 10 ml/dish of Met-Cys-free DMEM supplemented with 400 Ci each of³⁵S-methionine and 35S-cysteine (NEN Life Science Products, Boston,Mass.). Labeled cells were harvested using 5 mM EDTA in PBS, pelletedand frozen until needed.

[0188] To label the HIV-1 gp120 envelope glycoprotein, HEK-293T cells(American Type Culture Collection) grown to 70-80% confluence weretransfected (Geneporter transfection reagent, Gene Therapy Systems, SanDiego, Calif.) with plasmids expressing secreted gp120 from HIV-1strains ADA and HXBc2 (ref. Kolchinsky). Twenty-four hours after thetrnsfection, the medium was replaced with labeling medium, as describedabove. The cell supernatants containing ³⁵S-cysteine/methionine-labeledgp120 were harvested every 48 hours a total of three times. The labeledgp120 was purified from the pooled supernatants using a Protein ASepharose-F105 antibody column, as described (ref Wu et al).

[0189] Coating of Dynabeads by Antibodies and Streptavidin.

[0190] Tosylactivated Dynabeads® M-280 (Dynal, Inc., Lake Success, N.Y.)were conjugated with 1D4 antibodies (National Cell Culture Center,Minneapolis, Minn.), and streptavidin (Vector Laboratories, Inc.,Burlingame, Calif.) at a molar ratio 10:1 unless specifically mentioned.Approximately 6×10⁸ beads in 1 ml volume were vortexed, pelleted on amagnetic separator (Dynal) and resuspended in 1 ml of binding buffer(0.1 M sodium phosphate, pH 7.4) containing 1 mg of 1D4 antibody and 30Tg of streptavidin. After incubation on a rocking platform for 20 hoursat 37° C., the unbound surface reactive groups on the beads wereinactivated by treatment with 0.2 M Tris-HCI (pH 8.5) for 4 hours at 37°C. The noncovalently absorbed proteins were removed by a one-hourincubation in medium composed of 1% cyclohexyl-pentyl-ã-D-maltoside(Cymal™-5) detergent (Anatrace, Maumee, Ohio), 20 mM Tris-HCI (pH 7.5),100 mM (NH₄)₂SO₄ and 1M NaCI. Then the 1D4/streptavidin-beads werewashed twice and stored at 4° C. in PBS. The efficiency of antibodyconjugation to the beads, which was estimated by FACS using anti-mouseR-phycoerytrin-conjugated IgG (IgG-PE) (Boehringer Mannheim,Indianapolis, Ind.), was approximately 5×10⁴ antibody molecules/bead.The 2D7/Streptavidin conjugation was accomplished using the sameprotocol.

[0191] Preparation of Lipid Solutions for Liposomal MembraneReconstitution.

[0192] All lipids were obtained as chloroform solutions from AvantiPolar Lipids (Alabaster, Ala.). A total of 10 mg of chloroform-dissolvedlipids 1-Palmitoyl-2-Oleoyl-sn-Glycero-3-Phosphocholine (POPC),1-Palmitoyl-2-Oleoyl-sn-Glycero-3-Phosphoethanolamine (POPE) andDimyristoylphosphatidic acid (DMPA), mixed in a molar ratio of 6:3:1,were dried in a 2-ml polyethylene tube under a vacuum until all of thesolvent was removed. One milliliter of PBS was added to the tube and aliposomal solution was obtained by 1-2 min ultrasonication in an icebath using the Ultrasonic Processor (Heat Systems, Inc., Farmingdale,N.Y.). Liposomal solutions of total lipids from membranes of Cf2Thcells, which were extracted with chloroform/methanol (ref Folch), wereprepared similarly, using a final lipid concentration of 10 mg/ml.Liposomal solutions of the head group-modified synthetic lipidsdipalmitoylphosphoethanolamine-N-Biotinyl (Biotinyl-DPPE) anddioleoylphosphoethanolamine-Lissamine Rhodamine B (Rho-DOPE), at a finalconcentration of 1 mg/ml, were prepared separately using the sameprotocol. All liposomal solutions were kept in liquid N₂ until use.

[0193] Formation of Proteoliposomes with Purified CCR5.

[0194] Approximately 10⁸ Cf2Th/CCR5 cells were lysed in 10 ml ofsolubilization buffer (S-buffer) composed of 100 mM (NH₄)₂SO₄, 20 mMTris-HCI (pH 7.5), 10% Glycerol, 0.5% (w/v) Cymal™-5 and ProteaseInhibitor Mixture (one tablet of Complete™(Boehringer Mannheim) per 50ml) for 30 minutes at 4° C. Cell debris was removed by 30 mincentrifugation at 150,000×g. Approximately 5×10⁸ 1D4/Streptavidin-coatedbeads washed in S-buffer were added to the cleared cell lysate andincubated in it for 1 h at 4° C. on a rocking platform. The CCR5-boundbeads were then removed from the cell lysate and extensively washed inS-buffer. For formation of the lipid membrane around the CCR5-containingbeads, 1 mg of liposomes composed of either synthetic lipid mixtures orCf2Th cellular lipids was combined with 10 μg of liposomes made fromBiotinyl-DPPE and solubilized in 1 ml S-buffer. When fluorescentlabeling of the lipid membrane was desired, 10 μg of Rho-DOPE was addedto the mixture. This detergent-containing mixture was added toCCR5-containing beads and, after 1 hour incubation at 4° C., thedetergent was slowly removed by dialysis for 24 hours at 4° C. in12,000-kDa dialysis tubing against 100 mM (NH₄)₂SO₄, 20 mM Tris-HCI (pH7.5) and 10% glycerol. The excess of unbound lipid and residualdetergent was removed on a magnetic separator and proteoliposomes werestored in PBS at 4° C. for up to two months.

[0195] The protein composition of CCR5-proteoliposomes was analyzed bysilver staining or, when ³⁵S-cysteine/methionine-labeled CCR5 was used,by autoradiography. For these purposes, 10⁷ proteoliposomes wereresuspended in 2% SDS-sample buffer and, after 1 hour incubation at 55°C., the eluted sample was run on an 11% polyacrylamide mini-gel underreducing conditions.

[0196] Ligand Binding to CCR5-Proteoliposomes.

[0197] The binding of the 2D7 anti-CCR5 antibody was analyzed by FACSand confocal microscopy, using 2D7 conjugated with R-phycoerydirin(2D7-PE). CCR5-proteoliposomes were suspended in 5% BSA fetal calf serumin PBS or, in some experiments, in binding buffer (see below) andincubated with 2D7-PE for one hour at 22° C. The proteoliposomes werethen washed in the same buffer, fixed in 2% formaldehyde in PBS, andanalyzed by FACS or confocal microscopy.

[0198] The binding of the HIV-1 gp120 glycoprotein toCCR5-proteoliposomes was analyzed by FACS using unlabelled gp120 (JR-FLstrain) or by SDS-polyacrylamide gel analysis of bound, radiolabeledgp120 proteins. For the FACS analysis, CCR5-proteoliposomes weresuspended in 0.5 ml binding buffer (150 mM NaCl, 5 mM CaCl₂, 2 mM MgCl2,20 mM Tris, pH 7.5) and incubated for one hour at 22° C. with 3-5 {haeckover (s)}g JR-FL gp120 or with JR-FL gp120 that had been preincubatedfor one hour at 37° C. with an equimolar concentration of sCD4.Afterwards, the anti-gp120 antibody C11 (kindly provided by Dr. JamesRobinson, Tulane University) and a fluorescein-conjugated goatanti-human IgG (Pharmingen) were added, each at a final concentration of3-5 Tg/ml. Following incubation at 22° C. for one hour, theCCR5-proteoliposomes were washed in the binding buffer, fixed in 2%formaldehyde in PBS, and used for FACS and confocal microscopy.

[0199] For the studies of radiolabeled HIV-1 gp120 binding toCCR5-proteoliposomes, the metabolically labeled gp120 glycoproteins froma CCR5-using HIV-1 strain, ADA, and from a CXCR4using HIV-1 strain,HXBc2, were employed. In a preferred embodiment, one would use aradiolabelled HIV-1 gp120 proteoliposome to look at binding to the CCR5proteoliposomes. The gp120 glycoproteins were incubated in either thepresence or absence of sCD4 (10 nM final concentration) for one hour at37° C. Approximately 10⁷ CCR5-proteoliposomes were resuspended in 1 mlof binding buffer and incubated with the gp120 glycoproteins for 1 hourat 22° C. The proteoliposomes were extensively washed in the bindingbuffer and then resuspended in SDS-sample buffer containing5%-mercaptoethanol. After boiling for 2 minutes, the samples were loadedon 10% polyacrylamide mini-gels and analyzed by autoradiography.

[0200] Protein Composition of CCR5-Proteoliposomes

[0201] To examine the cellular proteins incorporated into theproteoliposomes, Cf2Th-CCR5 cells were metabolically labeled with³⁵S-cysteine and ³⁵S-methionine and used for proteoliposome formation.The proteoliposomes were incubated in SDS-sample buffer at 55° C. forone hour and the labeled proteins analyzed on polyacrylamide gels.Prominent bands associated with mature CCR5 (43 kDa) and a previouslyseen CCR5 derivative (36 kDa) were observed, as well as faint bandsassociated with higher-molecular weight aggregates of CCR5. Othercellular proteins were apparently present at only trace levels. Theseresults indicate that CCR5 is the major cellular protein in theproteoliposomes.

[0202] The proteins in the paramagnetic proteoliposomes were alsoexamined by silver staining of polyacrylamide gels of the SDS lysates.The only other bands visible in addition to the CCR5 bands describedabove were those associated with the 1D4 antibody heavy and light chains(55 and 25 KDa, respectively) and streptavidin (60 KDa) (data notshown). This demonstrates that apparently, no cellular proteins otherthan CCR5 are incorporated stoichiometrically into the paramagneticproteoliposomes.

[0203] Analysis of the Lipid Bilayer Membrane in CCR5-Proteoliposomes

[0204] The total quantity of lipid incorporated into the proteoliposomeswas determined. FACS analysis of CCR5-proteoliposomes formed withincreasing amounts of lipid containing 1% rhodamine-DOPE revealed thatapproximately 80-90 Tg of lipid was acquired per 10⁸ beads (FIG. 11).This is higher than the amount of lipid (approximately 40 Tg) that istheoretically needed to form bilayers surrounding beads of 2.8 Tmdiameter (see formula in FIG. 11, inset). This difference can beexplained by the irregularity of the bead surface, which was documentedby scanning electron microscopy (data not shown), and which couldcontribute to the formation of small micelle-like structures in thecrevasses of the bead surface. Additionally, some of the input lipid mayhave been lost during dialysis.

[0205] The CCR5-proteoliposomes were also studied by confocalmicroscopy. The control paramagnetic beads did not exhibit fluorescenceindicative of rhodamine-DOPE incorporation. By contrast, theCCR5-proteoliposomes that had been formed with 1% rhodamine-DOPEfluoresced intensely and uniformly. No lipid vesicles or otherstructures greater than 0.1 μm were observed on the surface of thefluorescently labeled CCR5-proteoliposomes. These data are consistentwith the CCR5-proteoliposomes being surrounded by a single lipid bilayermembrane with at most small irregularities.

[0206] Ligand Binding Properties of CCR5-Proteoliposomes

[0207] CCCR5-proteoliposomes efficiently bound the 2D7 antibody, whichrecognizes a conformation-dependent epitope on the CCR5 ectodomain.

[0208] To examine the ability of the CCR5-proteoliposomes to bind theHIV-1 exterior envelope glycoprotein, the gp120 glycoprotein from theCCR5-using strain JR-FL was preincubated with a soluble form of CD4(sCD4) to induce the high-affinity interaction with CCR5. The gp120/sCD4complex was incubated with CCR5-proteoliposomes, after which the boundcomplexes were detected by the C11 anti-gp120 antibody. Binding of thegp120 glycoprotein/sCD4 complexes to the CCR5-proteoliposomes, but notto control proteoliposomes lacking CCR5, was readily detected.

[0209] The binding of the HIV-1 gp120 glycoprotein to theCCR5-proteoliposomes was also examined in a different assay. Equivalentamounts of metabolically labeled gp120 glycoproteins from an HIV-1strain, HXBc2, which does not use the CCR5 protein as a coreceptor, andfrom the ADA strain, which uses CCR5 as a coreceptor, were added to theCCR5-proteoliposomes. Only the ADA gp120 glycoprotein detectably boundthe CCR5-proteoliposomes. This binding was enhanced by the addition ofsCD4. The binding of the ADA gp120/sCD4 complex to theCCR5-proteoliposomes was inhibited by preincubation of theproteoliposomes with the 2D7 anti-CCR5 antibody. These results indicatethat the gp120 glycoprotein from a CCR5-using HIV-1 specifically bindsCCR5 in the proteoliposome, and that CD4 binding enhances the gp120-CCR5interaction, as has been observed with cell surface CCR5 (Wu, L. et al.(1996) Nature 384: 179-183; Trkola, A. et al. (1996), Nature 384:184-187).

[0210] Stability of CCR5-Proteoliposomes

[0211] The effects of alterations in pH, ionic strength and temperatureon the stability of the CCR5-proteoliposomes were examined.Rhodamine-DOPE-labeled CCR5-proteoliposomes were exposed to acidic(pH=3) or basic (pH=10) conditions for 30 minutes, after which they werereturned to a neutral pH environment. The fluorescence intensitymeasured by FACS was comparable to that observed for untreated controlCCR5-proteoliposomes (data not shown). Fluorescence intensity was alsonot affected by incubation in solutions of different ionic strengths,ranging from less than 1 mM to 3M NaCl (data not shown). The binding ofthe 2D7 antibody to CCR5-proteoliposomes was completely disrupted byincubation of the antibody-proteoliposome complex at pH 3.0 for 30minutes. However, the ability of the 2D7 antibody to rebind theCCR5-proteoliposomes was completely restored by returning the pH to 7.0.The CCR5-proteoliposomes were stable at temperatures up to 50° C. forshort periods of time (less than two hours) and could be stored for atleast two months in PBS at 4° C. without loss of binding properties.

[0212] We have thus shown that an integral membrane protein such as theGPCR CCR5 can be expressed at reasonably high levels in mammalian cellsand purified in its native state in detergent-containing solutions. Wehave shown that the purified CCR5 can be reconstituted into a nativelipid membrane environment formed on the surface of paramagnetic beads.Accordingly, with minor adjustments, the approach is applicable to manyintegral membrane proteins.

Example 3

[0213] Proteoliposomes Containing CXCR4

[0214] Purification of CXCR4 Proteoliposomes

[0215] CXCR4-Cf2Th cells were grown to full confluency in 100 mm cellculture dishes. Cells were detached from the dish with 1×PBS/5mM EDTAand pelleted in microcentrifuge tubes at 1×10⁸ cells/pellet The pelletwas resuspended in an ice cold buffer containing 100 mM (NH4)2SO4, 20 mMTris pH 7.5, 20% glycerol, 1× Complete (Roche) protease inhibitorcocktail and 1% of either CHAPSO (Anatrace) or Cymal-7 (Anatrace).Resuspended cells were incubated for 5 minutes on ice followed by 25minutes at 4° C. on a Nutator (Fisher Scientific). After incubation,cell debris was removed by centrifugation at 14,000×g for 30 minutes at4° C. The supernatant was transferred to a new microcentrifuge tube and5×10⁸ 1D4 conjugated M-280 Dynal beads were added. Cell lysate wasincubated with beads for 2.5 hours at 4° C. on a Nutator. The tube wasthen placed in a Dynal MPC-S magnet to remove the beads. The beads werewashed two times with ice cold washing buffer (either 1% CHAPSO orCymal-7, 100 mM (NH4)₂SO₄, 20 mM Tris pH 7.5 and 20% glycerol). Afterwashing, beads prepared with CHAPSO were resuspended in 2.5 ml of icecold CHAPSO washing buffer containing 1.5 mg1-Palmitoyl-2-Oleoyl-sn-Glycero-3-Phosphocholine, 0.75 mg1-Palmitoyl-2-Oleoyl-sn-Glycero-3-Phosphoethanolamine, 0.225 mg 1.2Dioleoyl-sn-Glycero-3-Phosphate and 0.025 mgBiotinyl-Phosphoethanolamine. Cymal-7 prepared beads were resuspended inice cold 1% Cymal-5 washing buffer containing the above describedlipids. The solution was then injected into a Slide-A-Lyzer (Pierce, 10kDMWCO) and dialyzed for 24 hours against washing buffer containing nodetergent at 4° C. The samples were dialyzed in a specially designedmachine that constantly rotated the Slide-A-Lyzer to prevent settling ofthe beads. Following dialysis, the paramagnetic proteoliposomes wereremoved from the Slide-A-Lyzer and washed two times in 1×PBS/2% FBS toremove unbound lipid and any remaining detergent. Proteoliposomes werestored in 1×PBS/2% FBS/0.02% sodium azide for up to two months at 4° C.

[0216]FIG. 12 shows binding of the 12G5 antibody toCXCR4-proteoliposomes and to CXCR4-expressing cells.CXCR4-proteoliposomes were prepared as described in the text from cellsexpressing human CXCR4 with a C-terminal C9 tag. The binding of the 12G5antibody, which recognizes a conformation-dependent structure on CXCR4,to the CXCR4-expressing cells and CXCR4-proteoliposomes is shown. Theapparent affinity of the 12G5 antibody for the CXCR4 on theproteoliposome surface is at least as good as that for CXCR4 on cells. Asimilar result was obtained for the conformation-dependent,CXCR4-directed antibody FAB173 (data not shown).

[0217]FIG. 13 shows binding of SDF-1α to CXCR4 on cells andproteoliposomes. Radiolabeled SDF-1α, the natural CXCR4 ligand, wasincubated with either CXCR4-expressing cells or proteoliposomes bearingCXCR4 or CCR5. Unlabeled (cold) SDF-1α was added in increasing amounts,and the amount of radiolabeled SDF-1α bound to the cells orproteoliposomes was measured. The SDF-1α bound with high affinity to theCXCR4-expressing cells and CXCR4-proteoliposomes, but not to theCCR5-proteoliposomes.

Example 4

[0218] Screening a Phage Display Library with Proteoliposomes Containinggp160

[0219] The defined, reconstituted gp 160-proteoliposomes were used topan a highly complex, human single-chain antibody phage display librarygenerated in the laboratory of Dr. Wayne Marasco at the Dana-FarberCancer Institute. After four rounds of panning, 96 clones were analyzed;87 of the 96 clones were specific for gp120 as determined by ELISA (datanot shown). The 9 non-reactive clones may represent oligomer specificantibodies or irrelevant reactivities. To confirm that the isolatedphage possessed single-chain antibodies specific for gp160, FACSanalysis was performed on cells expressing gp160 comparing anti-gp120specific serum with the bacterial supernatant, an mouse anti-M13 phageIgG and anti-mouse-PE (FIG. 14). Further analysis of the soluble phagedisplayed single-chain antibodies is ongoing to determine theirspecificity. In any case, these intriguing preliminary data demonstratethe potential of the gp160-proteoliposomes to select and possibly elicitunique envelope-directed reactivities.

Example 5

[0220] Antibodies to Proteoliposomes Containing gp160

[0221] To confirm that the gp160 proteoliposomes could elicit envelopeglycoprotein-specific antibodies, Balb/c mice were immunized IP with5×10⁷ proteoliposomes. By gel analysis, we estimated that each mousereceived 1-2 μg of envelope glycoprotein per inoculation. To insure thatthe adjuvant would not disrupt the integrity of the reconstitutedmembrane, we preimmunized experimental mice IP with Ribi adjuvant 24hours prior to inoculation of the beads. Subsequently we have performedmembrane stability studies by incubation of rhodamine-DOPE-stained gp160proteoliposomes in Ribi adjuvant for 2 and 24 hours. The beads werevisualized by fluorescent microscopy and no decrease in therhodamine-DOPE signal was observed on beads exposed to adjuvant (datanot shown). Additional mice can be immunized with beads in Ribi adjuvantby various routes to optimize quantitative antibody responses.

[0222] For the initial study, 2 μg of monomeric YU2 gp120 in Ribiadjuvant was used as a positive control and membrane-reconstituted beadslacking gp160 glycoprotein were used as negative controls. After 3inoculations, we have detected anti-gp120 antibodies in the sera of themice from both the monomeric gp120 control group and the trimeric gp160bead group, but not from the sera of negative control mice (FIG. 15).This study demonstrates the feasibility of utilizing the trimeric gp160proteoliposomes as immunogens to better elicit neutralizing antibodiesor elicit trimer-specific antibodies that can be isolated andcharacterized by monoclonal analysis. Such reagents are invaluable toolsfor the further elucidation of HIV-1 envelope glycoprotein higher-orderstructure.

Example 6

[0223] Proteoliposomes Containing gp120-gp41 Complexes

[0224]FIG. 16 shows retention of HIV-1 gp120-gp41 association indetergent lysates. The envelope glycoproteins from the 89.6 HIV-1 straincontain a deletion of the gp41 cytoplasmic tail and are tagged at theC-terminus with the C9 peptide epitope, which is recognized by the 1D4antibody. The gp120-gp41 proteolytic cleavage site is intact in thisconstruct. 293T cells were transfected with a plasmid expressing the89.6 envelope glycoproteins, in some cases along with a plasmidexpressing furin (189-192). The cells were lysed in buffer containing 1%CHAPSO, and the lysates incubated with 1D4 antibody-coated beads. Afterwashing, the captured envelope glycoproteins were boiled off the beadsand resolved on a 6% SDS-polyacrylamide gel. The gel was Western blottedand developed with a rabbit anti-gp120 serum and HRP-conjugatedanti-rabbit IgG (left panel). The observed trimeric gp120 wasprecipitated by the 1D4 antibody through its association with gp41. Theblot was then stripped and reprobed with the 1D4 antibody andHRP-conjugated anti-mouse IgG (right panel). Note that, in this example,the coexpression of furin did not significantly increase the amount ofproteolytically processed envelope glycoproteins captured on the beadsurface, probably because proteolytic cleavage is already very efficientin this context.

Example 7

[0225] Env-Proteoliposomes Elicit HIV-1 Envelope Glycoprotein Antibodies

[0226]FIG. 17 shows immunization of mice with env-proteoliposomes. In apilot study, four mice per group were immunized intraperitoneally (primeplus two boosts) with gp120 (in Ribi adjuvant), with Env-proteoliposomesalone, or with Env-proteoliposomes in Ribi adjuvant. The ability of thesera to recognize gp120-coated ELISA plates is shown. The ELISA wasdeveloped with horseradish peroxidase-conjugated anti-mouse IgG andoptical density is indicated on the Y axis. Mean values are shown, andless than 30% deviation from the mean was observed in individualanimals. The results indicate that proteoliposomes can elicit antibodyresponses to HIV-1 envelope glycoproteins, and that Ribi adjuvant canenhance those responses.

[0227]FIG. 18 shows that sera from mice immunized withenv-proteoliposomes react efficiently with cell-surface HIV-1 envelopeglycoproteins. Four mice per group were immunized intraperi-toneallywith either Dynal beads coupled to the 1D4 antibody, Env-proteoliposomesalone, or Env-proteoliposomes in Ribi adjuvant. After priming and fourboosts, the sera were tested for the ability to stain cells expressingthe HIV-1 envelope glycoproteins of the same strain as the immunogen.The mean values for mean fluorescence intensity (MFI) are shown for eachgroup of mice.

[0228] All references described herein are incorporated by reference.

We claim:
 1. A stable immunogenic proteoliposome comprising: a sphericalor elliptoid shape having a ligand to an immunogenic transmembraneprotein anchored to the shape, wherein said shape's surface issurrounded by a lipid membrane; and an isolated integral membraneprotein bound to said ligand, wherein said integral membrane protein'stransmembrane domain(s) are in said lipid membrane, and wherein saidimmunogenic transmembrane protein is a multimer and has a wild-typeconformation.
 2. The stable immunogenic proteoliposome of claim 1,further comprising an attractant coating said shape's surface, whereinsaid lipid solution has a moiety that binds to the attractant forming alipid membrane surrounded shape.
 3. The stable immunogenicproteoliposome of claim 1 or 2, wherein the immunogenic transmembraneprotein is an envelope glycoprotein.
 4. The stable immunogenicproteoliposome of claim 2 or 3, wherein the attractant is streptavidinor avidin and the moiety in the lipid membrane is biotin.
 5. The stableimmunogenic proteoliposome of claim 4, wherein the ligand is anantibody.
 6. The stable immunogenic proteoliposome of claim 5, whereinthe lipid membrane is a lipid bilayer.
 7. The stable immunogenicproteoliposome of claim 6, wherein the envelope glycoprotein containstwo, three or four monomerric units.
 8. The immunogenic proteoliposomeof claim 7, wherein the envelope glycoprotein is a lentivirus envelopeglycoprotein.
 9. The immunogenic proteoliposome of claim 8, wherein thelentivirus envelope glycoprotein is from HIV-1 or HIV-2.
 10. Theimmunogenic proteoliposome of claim 9, wherein the envelope glycoproteinis a HIV-1 gp120 trimer, a HIV-1 gp160 trimer, or a trimeric fragmentthereof
 11. A method of obtaining an antibody to an envelopeglycoprotein comprising: screening a library of antibodies with theimmunogenic proteoliposome of claims 7, 8, 9 or 10 and selecting saidantibodies that bind to said proteoliposome.
 12. The method of claim 11,wherein the avidity of the binding is further determined.
 13. The methodof claim 11, wherein the antibody is an antibody to an HIV-1 envelopeglycoprotein.
 14. The method of claim 13, wherein the envelopeglycoprotein is the HIV-1 gp120 trimer, the HIV-1 gp160 trimer or thetrimeric immunogenic fragment thereof.
 15. A method of inducing animmunogenic response to an HIV-1 virion comprising administering aneffective amount of the immunogenic proteoliposome of claims 7, 8, 9 or10 to a human and at periodic times thereafter administering subsequentboosting amounts of said immunogenic proteoliposome or said immunogenicenvelope glycoprotein.
 16. A method of inducing an immunogenic responseto an HIV-1 virion comprising administering to a human the antibodyobtained by the method of claim 14.